Activation of Nrf2 by SF or CA improves metabolic disorder in an STZ-induced diabetic model.
The effectiveness of dietary Nrf2 activators in alleviating metabolic disorder was assessed in the STZ-induced diabetic model in both Nrf2+/+ and Nrf2−/− mice, and common metabolic disorder indices associated with diabetes were measured. In response to STZ, mice showed significantly increased blood glucose levels (), reduced weight gain (), increased urine production (), and increased water uptake (). Importantly, treatment of diabetic animals with SF or CA significantly alleviated all indices of metabolic dysfunction in Nrf2+/+ animals only, demonstrating the Nrf2-dependent response to SF and CA. In addition, STZ injection decreased insulin levels significantly, which was unaltered by treatment with SF or CA regardless of genotype (), indicating that the protective effects of SF and CA were not insulin dependent. Since treatment with Nrf2 activators began 2-weeks post-STZ injection, after the pancreatic β-cells had been destroyed and diabetes was established, we did not expect SF or CA treatments to influence circulating insulin levels in this study. Taken together, these results indicate that SF or CA treatment significantly attenuated metabolic disorder induced by STZ, and that this protective effect was independent of circulating insulin levels.
FIG. 1. Activation of Nrf2 by SF or CA improves metabolic disorder in an STZ-induced diabetic model. A: The average level of blood glucose in each animal group is plotted. STZ animals (■) showed significant increase in blood glucose levels in both Nrf2 (more ...)
SF or CA alleviates renal damage induced in the STZ diabetic model.
To investigate the therapeutic effects of Nrf2 activation on improvement of kidney performance in the STZ diabetic model, functional and pathological changes of the kidney were measured. The ratio of kidney weight-to-body weight was higher in all STZ-injected groups compared with untreated, which was significantly attenuated by treatment with SF or CA in Nrf2+/+ mice (). Next, as indices of albuminuria, both urinary albumin excretion (UAE) and the urine albumin-to-creatinine ratio (UACR) were determined at 0, 10, and 18 weeks. Injection with STZ increased UAE and UACR in all treated groups; however, this increase was blunted by SF or CA treatment in Nrf2+/+ mice but not Nrf2−/− mice (). These results demonstrate that SF or CA is able to improve albuminuria in Nrf2+/+ diabetic mice.
FIG. 2. SF or CA alleviates renal damage induced in the STZ-diabetic model. A: Kidney-to-body weight ratios are provided. STZ injection increased weight ratios in both Nrf2+/+ (top panel) and Nrf2−/− (bottom panel) animals, which could be significantly (more ...)
In agreement with the urinalysis, SF or CA treatment alleviated the pathological alterations of the glomerulus as revealed by histological examination. Glomerular lesions, including K-W (Kimmelstiel Wilson) nodules, were observed in HE-stained tissue sections from STZ-injected mice (, HE panel, arrows). Treatment with SF or CA2 effectively restored the normal morphology of glomeruli in Nrf2+/+ but not Nrf2−/− mice (, HE panel). Glycogen deposition in the glomeruli was measured by PAS staining and scored to indicate the severity of glomerulosclerosis. PAS staining showed STZ-induced glomerulosclerosis, which was significantly improved in SF- or CA2-treated Nrf2+/+ but not Nrf2−/− mice (, PAS panel, and ). Since ECM deposition is another hallmark of glomerulosclerosis, collagen was visualized using Masson’s trichrome staining. STZ treatment resulted in collagen accumulation inside glomeruli or in the periglomerular area, which was reduced in SF- or CA-treated Nrf2+/+ but not Nrf2−/− groups (, trichrome panel). As an independent criterion for glomerulosclerosis, thickening of the GBM was measured by electron microscopy (). STZ injection increased thickness of the GBM, which was reverted to control by SF or CA treatment only in Nrf2+/+ mice (). These results clearly demonstrate the ability of SF and CA treatment to attenuate STZ-induced pathological alterations both in renal function and structure during the progression of diabetic nephropathy.
SF- or CA-induced activation of the Nrf2 pathway confers protection against renal oxidative damage.
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 (). 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 (). 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.
FIG. 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. (more ...)
Activation of Nrf2 reduces TGF-β1, ECM deposition, and p21 expression.
Given that treatment with SF or CA diminished renal disorders resulting from STZ-induced diabetes, the molecular mechanisms underlying Nrf2-dependent protection of the kidney by SF or CA were explored. Previously, our laboratory reported a negative association between Nrf2 and TGF-β1. In accordance with that report, the basal expression of TGF-β1 was higher in Nrf2−/− mice compared with Nrf2+/+ mice (). Diabetes induced by STZ significantly upregulated protein levels of TGF-β1 and its downstream effectors, FN, collagen IV, and p21, which were significantly abrogated by treatment with SF or CA in the Nrf2+/+ animals only (). Treatment with SF and CA in the Nrf2−/− mice did not alter TGF-β1, ECM, or p21, indicating that the negative regulation of TGF-β1 and subsequent downstream effectors (ECM and p21) observed with SF and CA treatment is Nrf2 dependent.
FIG. 4. Activation of Nrf2 reduces TGF-β1, ECM deposition, and p21 expression. A: Immunoblot analysis was performed on lysates from whole kidney (left panel), and band intensities were quantified (right panel, bar graphs) and reported as relative expression (more ...)
Activation of Nrf2 diminishes mesangial ROS generation under hyperglycemic conditions.
Mesangial cells play a crucial role in dictating the function of glomeruli. To understand the molecular mechanism(s) by which activation of Nrf2 is able to preserve renal function during the progression of diabetic nephropathy, cultured primary HRMCs were used. HRMCs growing in NG (5.5 mmol/L) media were shifted to either NG (plus 19.5 mmol/L mannitol) or HG (25 mmol/L) media to mimic hyperglycemic conditions in the presence or absence of an Nrf2 activator. Similar to the results reported above in the animal model of STZ-induced diabetes, culturing HRMCs under hyperglycemic conditions also activated the Nrf2 pathway (), presumably through oxidative stress.
FIG. 5. Activation of Nrf2 diminishes mesangial ROS generation under hyperglycemic conditions. A: Nrf2 expression and localization were assessed in HRMCs incubated in NG, HG, HG+tBHQ, HG+SF, or HG+CA DMEM media for 48 h. Incubation in HG as well as treatment (more ...)
To support the in vivo findings reported above, HRMCs grown in HG media were treated with SF, CA, and tBHQ, a well-characterized Nrf2 activator. As expected, higher Nrf2 expression and predominate nuclear localization of Nrf2 were observed in response to tBHQ, SF, or CA treatment, indicating activation of the Nrf2 pathway (). Consistently, protein levels of Nrf2 were enhanced after 48-h incubation in HG media and further induced by tBHQ, SF, or CA treatment (). Similarly, protein levels of Nrf2 downstream targets including NQO1, GCS, or Mrp2, were increased in response to HG and further enhanced by treatment with an Nrf2 activator (). To test if HRMCs growing in HG media were under oxidative stress, the level of ROS was measured. Indeed, HRMCs growing in HG media had ROS levels approximately 3.5-fold higher compared with HRMCs growing in NG media (). As predicted, activation of Nrf2 by tBHQ, SF, or CA reduced ROS levels significantly (). These results demonstrate ROS generation as a likely source of mesangial cell damage in diabetes and further demonstrate the beneficial effects of Nrf2 pathway activation in combating oxidative stress.
High glucose–mediated mesangial cell growth inhibition and hypertrophy can be reversed by activation of Nrf2.
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 (). 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 (). 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 (), 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 (). 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 (). Collectively, these results demonstrate that hyperglycemia induced cell growth inhibition, and hypertrophy can be attenuated by activation of the Nrf2 pathway.
FIG. 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; (more ...)
Blockage of TGF-β1 by Nrf2 activation alleviates ECM production and p21-mediated mesangial cell growth inhibition and hypertrophy.
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 (). 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 (), while control siRNA had no effects on HRMC growth (). Furthermore, hypertrophy of HRMCs growing in HG was no longer observed when expression of p21 was reduced by siRNA (), indicating p21 controls mesangial growth inhibition and hypertrophy.
FIG. 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 (more ...)
To further confirm the negative regulation of Nrf2 on TGF-β1 and its downstream response, endogenous Nrf2 levels were modulated either by Nrf2-siRNA or by siRNA to Keap1, a negative regulator of Nrf2. As expected, Nrf2 levels were reduced by Nrf2-siRNA and increased by Keap1-siRNA (). The resulting effects of Nrf2 modulation display a negative Nrf2 regulation of TGF-β1 and its downstream effectors including FN, collagen IV, and p21. Specifically, downregulation of Nrf2 resulted in enhanced expression of these proteins, whereas upregulation of Nrf2 reduced expression of the TGF-β1 pathway under both NG and HG media (). Taken together, these results suggest that Nrf2 activation is able to confer renal protection in diabetic conditions through blockage of the TGF-β1 downstream response, specifically by reducing ECM overproduction and p21-mediated growth inhibition and hypertrophy.