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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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Blockage of TGF-β1 by Nrf2 activation alleviates ECM production and p21-mediated mesangial cell growth inhibition and hypertrophy. A: Expression of TGF-β1 and downstream proteins was assessed by immunoblot analysis in HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h (upper panel). Relative protein expression averaged over triplicate experiments are presented in bar graphs (lower panel). HG increased TGF-β1, FN, collagen IV, and p21. Coculture with an Nrf2 activator prevented increase of these proteins. *P < 0.05 compared to NG group. #P < 0.05 Nrf2 activators compared to HG alone (bar graph). B: Representative immunoblots and live cell growth curves from HRMCs transfected with p21-siRNA. Knockdown of p21 rescues growth inhibition induced by HG conditions. C: Representative images from flow cytometry analysis of forward light scatter analysis. Knockdown of p21 reduces HG-induced hypertrophy in HRMCs. D: Representative immunoblots from HRMCs transfected with control siRNA, Nrf2-siRNA, or Keap1-siRNA. Knockdown of Keap1 and Nrf2 illustrate the Nrf2-dependent repression of TGF-β1, ECM proteins, and p21 under both normal and hyperglycemic conditions. 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 7: Blockage of TGF-β1 by Nrf2 activation alleviates ECM production and p21-mediated mesangial cell growth inhibition and hypertrophy. A: Expression of TGF-β1 and downstream proteins was assessed by immunoblot analysis in HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h (upper panel). Relative protein expression averaged over triplicate experiments are presented in bar graphs (lower panel). HG increased TGF-β1, FN, collagen IV, and p21. Coculture with an Nrf2 activator prevented increase of these proteins. *P < 0.05 compared to NG group. #P < 0.05 Nrf2 activators compared to HG alone (bar graph). B: Representative immunoblots and live cell growth curves from HRMCs transfected with p21-siRNA. Knockdown of p21 rescues growth inhibition induced by HG conditions. C: Representative images from flow cytometry analysis of forward light scatter analysis. Knockdown of p21 reduces HG-induced hypertrophy in HRMCs. D: Representative immunoblots from HRMCs transfected with control siRNA, Nrf2-siRNA, or Keap1-siRNA. Knockdown of Keap1 and Nrf2 illustrate the Nrf2-dependent repression of TGF-β1, ECM proteins, and p21 under both normal and hyperglycemic conditions. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)

Mentions: To gain molecular understanding of how Nrf2 activation relieves HRMC hypertrophy and growth inhibition under hyperglycemic conditions, the expression of TGF-β1 and its downstream effectors were analyzed. Consistent with our in vivo data, hyperglycemia upregulated TGF-β1, FN, collagen IV, and p21 in HRMCs, which was suppressed by treatment with tBHQ, SF, or CA (Fig. 7A). Next, the functional contribution of the cell cycle regulator, p21, in mesangial cell growth inhibition and hypertrophy was investigated. Knockdown of p21 expression by transient transfection with p21-siRNA reduced the protein level of p21 and recovered the growth rate of HRMCs in HG media to that of NG conditions (Fig. 7B), while control siRNA had no effects on HRMC growth (Fig. 7B). Furthermore, hypertrophy of HRMCs growing in HG was no longer observed when expression of p21 was reduced by siRNA (Fig. 7C), indicating p21 controls mesangial growth inhibition and hypertrophy.


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)

Blockage of TGF-β1 by Nrf2 activation alleviates ECM production and p21-mediated mesangial cell growth inhibition and hypertrophy. A: Expression of TGF-β1 and downstream proteins was assessed by immunoblot analysis in HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h (upper panel). Relative protein expression averaged over triplicate experiments are presented in bar graphs (lower panel). HG increased TGF-β1, FN, collagen IV, and p21. Coculture with an Nrf2 activator prevented increase of these proteins. *P < 0.05 compared to NG group. #P < 0.05 Nrf2 activators compared to HG alone (bar graph). B: Representative immunoblots and live cell growth curves from HRMCs transfected with p21-siRNA. Knockdown of p21 rescues growth inhibition induced by HG conditions. C: Representative images from flow cytometry analysis of forward light scatter analysis. Knockdown of p21 reduces HG-induced hypertrophy in HRMCs. D: Representative immunoblots from HRMCs transfected with control siRNA, Nrf2-siRNA, or Keap1-siRNA. Knockdown of Keap1 and Nrf2 illustrate the Nrf2-dependent repression of TGF-β1, ECM proteins, and p21 under both normal and hyperglycemic conditions. 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 7: Blockage of TGF-β1 by Nrf2 activation alleviates ECM production and p21-mediated mesangial cell growth inhibition and hypertrophy. A: Expression of TGF-β1 and downstream proteins was assessed by immunoblot analysis in HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h (upper panel). Relative protein expression averaged over triplicate experiments are presented in bar graphs (lower panel). HG increased TGF-β1, FN, collagen IV, and p21. Coculture with an Nrf2 activator prevented increase of these proteins. *P < 0.05 compared to NG group. #P < 0.05 Nrf2 activators compared to HG alone (bar graph). B: Representative immunoblots and live cell growth curves from HRMCs transfected with p21-siRNA. Knockdown of p21 rescues growth inhibition induced by HG conditions. C: Representative images from flow cytometry analysis of forward light scatter analysis. Knockdown of p21 reduces HG-induced hypertrophy in HRMCs. D: Representative immunoblots from HRMCs transfected with control siRNA, Nrf2-siRNA, or Keap1-siRNA. Knockdown of Keap1 and Nrf2 illustrate the Nrf2-dependent repression of TGF-β1, ECM proteins, and p21 under both normal and hyperglycemic conditions. FSC-H, forward scatter. SSC-H, side scatter. (A high-quality color representation of this figure is available in the online issue.)
Mentions: To gain molecular understanding of how Nrf2 activation relieves HRMC hypertrophy and growth inhibition under hyperglycemic conditions, the expression of TGF-β1 and its downstream effectors were analyzed. Consistent with our in vivo data, hyperglycemia upregulated TGF-β1, FN, collagen IV, and p21 in HRMCs, which was suppressed by treatment with tBHQ, SF, or CA (Fig. 7A). Next, the functional contribution of the cell cycle regulator, p21, in mesangial cell growth inhibition and hypertrophy was investigated. Knockdown of p21 expression by transient transfection with p21-siRNA reduced the protein level of p21 and recovered the growth rate of HRMCs in HG media to that of NG conditions (Fig. 7B), while control siRNA had no effects on HRMC growth (Fig. 7B). Furthermore, hypertrophy of HRMCs growing in HG was no longer observed when expression of p21 was reduced by siRNA (Fig. 7C), indicating p21 controls mesangial growth inhibition and hypertrophy.

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