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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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SF- or CA-induced activation of the Nrf2 pathway confers protection against renal oxidative damage. A: Whole kidney lysates from three mice per group were subjected to immunoblot analysis with antibodies against Nrf2, NQO1, γ-GCS, and β-actin. The experiments were repeated three times, and one representative blot is shown (left panel). The intensity of bands from replicate immunoblots was quantified and plotted (right panel, bar graphs). *P < 0.05 compared with control. B: Fixed kidney tissues were stained with antibodies against Nrf2, NQO1, and γ-GCS. Representative images from each group showed a slight induction of Nrf2 and downstream targets upon STZ injection. Treatment with SF and CA further induced the Nrf2 pathway in Nrf2+/+ only. C: 8-oxo-dG staining assessed oxidative damage in the kidney, and a representative image is provided. D: Urine collected at the last 24 h of the experiment was used for measurement of urinary 8-oxo-dG. STZ injection caused significant DNA damage that was attenuated by treatment with SF and CA in Nrf2+/+ only. Data are expressed as mean ± SD (n = 3). *P < 0.05 compared with control. #P < 0.05 dietary treatment compared with STZ alone. (A high-quality color representation of this figure is available in the online issue.)
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Figure 3: SF- or CA-induced activation of the Nrf2 pathway confers protection against renal oxidative damage. A: Whole kidney lysates from three mice per group were subjected to immunoblot analysis with antibodies against Nrf2, NQO1, γ-GCS, and β-actin. The experiments were repeated three times, and one representative blot is shown (left panel). The intensity of bands from replicate immunoblots was quantified and plotted (right panel, bar graphs). *P < 0.05 compared with control. B: Fixed kidney tissues were stained with antibodies against Nrf2, NQO1, and γ-GCS. Representative images from each group showed a slight induction of Nrf2 and downstream targets upon STZ injection. Treatment with SF and CA further induced the Nrf2 pathway in Nrf2+/+ only. C: 8-oxo-dG staining assessed oxidative damage in the kidney, and a representative image is provided. D: Urine collected at the last 24 h of the experiment was used for measurement of urinary 8-oxo-dG. STZ injection caused significant DNA damage that was attenuated by treatment with SF and CA in Nrf2+/+ only. Data are expressed as mean ± SD (n = 3). *P < 0.05 compared with control. #P < 0.05 dietary treatment compared with STZ alone. (A high-quality color representation of this figure is available in the online issue.)

Mentions: Next, activation of Nrf2, its downstream targets, and oxidative damage in the kidney were assessed to demonstrate that the beneficial effect of SF or CA against renal damage is attributed to activation of the Nrf2 pathway and thus reduction of oxidative damage. Expression of Nrf2 and Nrf2 downstream targets NQO1 and γ-GCS was slightly increased after STZ injection, indicating induction of the Nrf2 pathway by renal oxidative stress. As expected, treatment with SF and CA markedly increased the protein levels of Nrf2, NQO1, and γ-GCS (Fig. 3A and B). Importantly, SF or CA treatment significantly reduced oxidative damage in the Nrf2+/+ kidney, as measured by the formation of 8-oxo-dG either locally in glomerular tissues or systemically from the urine (Fig. 3C and D). Treatment with SF or CA did not increase NQO1 or γ-GCS or reduce oxidative damage in Nrf2−/− mice. Collectively, these results indicate that the kidney of STZ-induced diabetic mice is under oxidative stress, and that the renal protection mediated by SF or CA in the diabetic model is, at least in part, derived from specific activation of the Nrf2-mediated antioxidant response and reduction of oxidative damage.


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)

SF- or CA-induced activation of the Nrf2 pathway confers protection against renal oxidative damage. A: Whole kidney lysates from three mice per group were subjected to immunoblot analysis with antibodies against Nrf2, NQO1, γ-GCS, and β-actin. The experiments were repeated three times, and one representative blot is shown (left panel). The intensity of bands from replicate immunoblots was quantified and plotted (right panel, bar graphs). *P < 0.05 compared with control. B: Fixed kidney tissues were stained with antibodies against Nrf2, NQO1, and γ-GCS. Representative images from each group showed a slight induction of Nrf2 and downstream targets upon STZ injection. Treatment with SF and CA further induced the Nrf2 pathway in Nrf2+/+ only. C: 8-oxo-dG staining assessed oxidative damage in the kidney, and a representative image is provided. D: Urine collected at the last 24 h of the experiment was used for measurement of urinary 8-oxo-dG. STZ injection caused significant DNA damage that was attenuated by treatment with SF and CA in Nrf2+/+ only. Data are expressed as mean ± SD (n = 3). *P < 0.05 compared with control. #P < 0.05 dietary treatment compared with STZ alone. (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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Figure 3: SF- or CA-induced activation of the Nrf2 pathway confers protection against renal oxidative damage. A: Whole kidney lysates from three mice per group were subjected to immunoblot analysis with antibodies against Nrf2, NQO1, γ-GCS, and β-actin. The experiments were repeated three times, and one representative blot is shown (left panel). The intensity of bands from replicate immunoblots was quantified and plotted (right panel, bar graphs). *P < 0.05 compared with control. B: Fixed kidney tissues were stained with antibodies against Nrf2, NQO1, and γ-GCS. Representative images from each group showed a slight induction of Nrf2 and downstream targets upon STZ injection. Treatment with SF and CA further induced the Nrf2 pathway in Nrf2+/+ only. C: 8-oxo-dG staining assessed oxidative damage in the kidney, and a representative image is provided. D: Urine collected at the last 24 h of the experiment was used for measurement of urinary 8-oxo-dG. STZ injection caused significant DNA damage that was attenuated by treatment with SF and CA in Nrf2+/+ only. Data are expressed as mean ± SD (n = 3). *P < 0.05 compared with control. #P < 0.05 dietary treatment compared with STZ alone. (A high-quality color representation of this figure is available in the online issue.)
Mentions: Next, activation of Nrf2, its downstream targets, and oxidative damage in the kidney were assessed to demonstrate that the beneficial effect of SF or CA against renal damage is attributed to activation of the Nrf2 pathway and thus reduction of oxidative damage. Expression of Nrf2 and Nrf2 downstream targets NQO1 and γ-GCS was slightly increased after STZ injection, indicating induction of the Nrf2 pathway by renal oxidative stress. As expected, treatment with SF and CA markedly increased the protein levels of Nrf2, NQO1, and γ-GCS (Fig. 3A and B). Importantly, SF or CA treatment significantly reduced oxidative damage in the Nrf2+/+ kidney, as measured by the formation of 8-oxo-dG either locally in glomerular tissues or systemically from the urine (Fig. 3C and D). Treatment with SF or CA did not increase NQO1 or γ-GCS or reduce oxidative damage in Nrf2−/− mice. Collectively, these results indicate that the kidney of STZ-induced diabetic mice is under oxidative stress, and that the renal protection mediated by SF or CA in the diabetic model is, at least in part, derived from specific activation of the Nrf2-mediated antioxidant response and reduction of oxidative damage.

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