Download Derivative of Bardoxolone Methyl, dh404, in an Inverse

Diabetes Volume 63, September 2014
3091
Sih Min Tan,1 Arpeeta Sharma,1 Nada Stefanovic,1 Derek Y.C. Yuen,1 Tom C. Karagiannis,2
Colin Meyer,3 Keith W. Ward,3 Mark E. Cooper,1 and Judy B. de Haan1
Derivative of Bardoxolone
Methyl, dh404, in an Inverse
Dose-Dependent Manner
Lessens Diabetes-Associated
Atherosclerosis and Improves
Diabetic Kidney Disease
Diabetes 2014;63:3091–3103 | DOI: 10.2337/db13-1743
collagen I, and proinflammatory interleukin-6. Higher
doses of dh404 were associated with increased expression of proinflammatory mediators MCP-1 and nuclear
factor-kB. These findings suggest that this class of
compound is worthy of further study to lessen diabetes
complications but that dosage needs consideration.
1Oxidative Stress Laboratory, Diabetic Complications Division, Baker IDI Heart and
Diabetes Institute, Melbourne, Australia
2Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Melbourne,
Australia
3Reata Pharmaceuticals, Inc., Irving, TX
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1743/-/DC1.
Corresponding author: Judy B. de Haan, [email protected].
See accompanying article, p. 2904.
Received 14 November 2013 and accepted 10 April 2014.
Diabetes is a common risk factor for both chronic kidney
disease and atherosclerosis (1,2). Despite the use of conventional therapies that include blood pressure, glucoselowering, and hyperlipidemic treatments, significant
numbers of diabetic patients suffer morbidity or mortality as a consequence of cardiovascular complications
and/or progress to end-stage renal failure. Novel interventions are needed to satisfy this unmet clinical need to
limit multiple diabetes-associated complications.
Evidence strongly supports a role for oxidative stress
and inflammation as underlying pathogenic mechanisms of
both diabetic renal and atherogenic complications. With
oxidative stress and inflammation now recognized to be
inextricably linked (3), approaches that limit oxidative
stress are likely to translate to reduced inflammation. Limiting oxidative stress by bolstering antioxidant defenses is
a novel alternative approach to antioxidant therapy that
© 2014 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
COMPLICATIONS
Oxidative stress and inflammation are inextricably linked
and play essential roles in the initiation and progression
of diabetes complications such as diabetes-associated
atherosclerosis and nephropathy. Bolstering antioxidant
defenses is an important mechanism to lessen oxidative
stress and inflammation. In this study, we have used a
novel analog of the NFE2-related factor 2 (Nrf2) agonist
bardoxolone methyl, dh404, to investigate its effects on
diabetic macrovascular and renal injury in streptozotocininduced diabetic apolipoprotein E2/2 mice. We show that
dh404, at lower but not higher doses, significantly lessens diabetes-associated atherosclerosis with reductions in oxidative stress (in plasma, urine, and vascular
tissue) and proinflammatory mediators tumor necrosis
factor-a, intracellular adhesion molecule-1, vascular cell
adhesion molecule-1, and monocyte chemotactic protein-1
(MCP-1). We demonstrate that dh404 attenuates functional (urinary albumin-to-creatinine ratio) and structural
(mesangial expansion) glomerular injury and improves
renal tubular injury. Liver functional and structural studies showed that dh404 is well tolerated. Complementary
in vitro studies in normal rat kidney cells showed that
dh404 significantly upregulates Nrf2-responsive genes,
heme oxygenase-1, NAD(P)H quinone oxidoreductase 1,
and glutathione-S transferase, with inhibition of transforming growth factor-b–mediated profibrotic fibronectin,
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dh404 Improves End-Organ Injury in Diabetic Mice
may yield more efficacious outcomes than standard vitamin
therapy (4). One approach that has attracted significant
attention is the augmentation of antioxidant defenses via
an upregulation of the NFE2-related factor 2 (Nrf2)/Kelchlike ECH-associated protein 1 (Keap1) pathway (5–9).
Numerous small-molecule activators of the Nrf2/
Keap1 pathway have been identified (10,11), including
bardoxolone methyl (BM), also known as CDDO-methyl,
a potent synthetic triterpenoid inducer of the phase 2
response (12,13). BM and other related antioxidant inflammation modulators (AIMs) such as CDDO-imidazole
(RTA 403) have demonstrated structural and functional
improvements in several rodent models of renal disease
(14–16), along with an increase in protective Nrf2, peroxisome proliferator–activated receptor g, and heme
oxygenase-1 (HO-1) genes (16). Given the known negative effects of oxidants and inflammation on the vasculature and the promising antioxidant, anti-inflammatory
and tissue-protective effects of AIM compounds (13), we
hypothesized that Nrf2 agonists may additionally protect
the diabetic vasculature against accelerated atherosclerosis. Our hypothesis is strengthened by recent data showing an adaptive induction of Nrf2-driven antioxidant
genes in response to hyperglycemia in human endothelial
cells (17) and the use of the Nrf2 activator sulforaphane
to induce Nrf2 activation of downstream target genes in
endothelial cells (18).
Interest in BM was further enhanced by two recent
phase 2 clinical trials in which this compound showed
a dose-dependent increase in the estimated glomerular
filtration rate (eGFR) initially after 56 days of treatment
(19) and in a second study (BEAM) (20) after 24 weeks of
treatment in patients with advanced chronic kidney disease and type 2 diabetes. In the latter study, the positive
effects of drug treatment persisted at 52 weeks after removal of the drug at 24 weeks. However, issues of drug
toxicity and off-target effects of this drug class have been
raised, particularly after the larger phase 3 clinical trial
(BEACON) with primary end points of time–to–first
occurrence of either end-stage renal disease or cardiovascular death (21) was terminated prematurely due to
unexplained adverse cardiovascular events. Recently, the
results of BEACON have been published and suggest a potential effect of BM on heart failure, further emphasizing
that this drug class needs more extensive investigations
with respect to not only renal but also cardiovascular end
points (22). Potential problems with toxicity and a worsening of diabetic nephropathy were also raised in a preclinical study examining the effects of BM analogs RTA
405 and dh404 on renal outcomes in diabetic Zucker fat
rats (23). The issue of toxicity was subsequently addressed
in a follow-up study (24) that showed that RTA 405 and
dh404 are well tolerated and did not confirm the previous
report of hepatic and renal toxicity.
To further clarify the effects of Nrf2 activation on diabetic
renal injury and investigate its potential role in lessening diabetes-associated atherosclerosis, in this study, we
Diabetes Volume 63, September 2014
treated diabetic apolipoprotein E–knockout (ApoE2/2) mice
with the BM analog dh404 for 18 weeks and assessed parameters associated with renal structural and functional injury as
well as atherosclerosis. As rodents metabolize BM to a metabolite that itself is not toxic to higher mammals but is
quite toxic to rodents, BM itself cannot be used for chronic
rodent studies. Therefore, tool compounds, including dh404
(also known as dh-CDDO-trifluoroethyl amide; see Supplementary Fig. 1), are used to probe rodent pharmacology for
this class and inform on the likely clinical behavior of BM.
Indeed, BM and dh404 exhibit very similar qualitative
biological properties (25) and the same mechanism of action,
in which dh404 has been shown to activate Nrf2 by preventing its degradation as a result of interrupting the ability of
Keap1 to bind to Nrf2. Therefore, dh404 is an appropriate
surrogate for studying this class of compound. We now present evidence, for the first time, that dh404 lowers oxidative
stress and proinflammatory mediators and protects against
diabetes-associated atherosclerosis in a dose-dependent
manner. In addition, dh404 improves renal structural and
functional injury while showing no detrimental effect on
liver function in this diabetic model.
RESEARCH DESIGN AND METHODS
BM Derivative dh404
dh404, solubilized in sesame oil (SO; Spectrum Chemical) at
3, 10, or 20 mg/kg body weight, was administered to mice
once per day by oral gavage.
Animals
Eight-week-old male C57Bl/J6 ApoE2/2 mice purchased
from the Animal Resource Centre (Perth, Australia) were
rendered diabetic by two intraperitoneal injections of
streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) at
100 mg/kg/day on 2 consecutive days (26–28).
In a first cohort, 10-week-old diabetic mice were
gavaged with either SO or 10 or 20 mg/kg dh404.
Additionally, a group of nondiabetic mice received
20 mg/kg dh404. Aortas and kidneys were collected
for quantitative RT-PCR (qRT-PCR) after 5 weeks of
gavage. A second cohort of diabetic mice was gavaged
with SO or 3, 10, or 20 mg/kg dh404 for 18 weeks.
Additionally, sham-injected nondiabetic mice were gavaged
with 20 mg/kg dh404. Weekly blood glucose, body weight,
and blood pressure measurements [using noninvasive
tail-cuff plethysmography (29)] were performed. Three
days prior to termination, urine was collected in 24-h
metabolic cages. Mice were killed by lethal injection of
2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO)
and direct puncture of the right ventricle to obtain blood.
Plasma was stored at 280°C. Plasma and urine were
analyzed for plasma alanine transaminase (ALT), aspartate transaminase (AST), and urine creatinine (Australian Specialized Animal Pathology Laboratory, Mulgrave,
Victoria, Australia). Aortas, kidney, and livers were fixed
in 10% neutral buffered formalin. Kidney cortex was snap
frozen in liquid nitrogen for RNA extraction.
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Evaluation of Atherosclerotic Lesion
qRT-PCR
En face analysis of lesions was conducted after staining
with Sudan IV Herxheimer’s solution (26–28). Plaque area
was calculated as the proportion of aortic intimal surface
area occupied by red-stained plaque (Adobe Photoshop
v6.0.1; Adobe Systems, New South Wales, Australia).
Lesions within the sinus were visualized after staining
with Oil Red O and quantitated as described previously
(26–28).
RNA from kidney and aorta was extracted using TRIzol
Reagent (Invitrogen) and cDNA synthesized as described
previously (27,28). Gene expression was determined after
real-time quantitative RT-PCR and analyzed as described
previously (27,28). Gene expression was normalized relative to 18S ribosomal RNA. Primers and probes are shown
in Supplementary Table 1.
Kidney Function Assessment
Urinary albumin-to-creatinine ratio was measured using
a mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX). Plasma cystatin C was measured using a
mouse cystatin C ELISA kit (30) (BioVendor Laboratory
Medicine, Brno, Czech Republic).
Oxidative Stress Assessment
Urinary 8-isoprostane and 8-hydroxy-2-deoxyguanosine
(8-OHdG) were assessed using commercially available EIA
kits (Cayman Chemical, Ann Arbor, MI, and StressMarq
Biosciences Inc, Victoria, BC, Canada, respectively).
Measures were expressed relative to urinary creatinine.
Plasma was analyzed for diamicron reactive oxygen
metabolites (dROMs), particularly hydroperoxides, using a Free Radical Analytical System-4 (H&D srl, Parma,
Italy) and expressed in Carratelli units (31).
In Vitro Experiments in Normal Rat Kidney Cells
Tubular NRK52E cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM containing 10% serum and 25 mmol/L glucose. Cells were
serum starved and pretreated with dh404 at 0.25, 0.5,
or 0.75 mmol or vehicle (DMSO) for 4 h before stimulation with 10 ng/mL transforming growth factor-b1
(TGF-b1) for 72 h (34). Treatment at $1 mmol dh404
for 48 h resulted in significant cell death (Supplementary
Fig. 2). Cells were collected for qRT-PCR or Western blot
analysis (28).
Statistical Analyses
Histological Assessment of Kidney
Data are expressed as mean 6 SEM and analyzed by
one-way ANOVA with Newman-Keuls post hoc testing.
Statistical analyses were performed using GraphPad
Prism version 6.0 (GraphPad, La Jolla, CA). A P value
,0.05 was considered statistically significant.
Mesangial and Tubulointerstitial Area
RESULTS
Kidney sections were stained with periodic acid-Schiff
(PAS). Mesangial area was quantitated using Image-Pro
Plus v6.0 (Media Cybernetics, Rockville, MD) and expressed
as percentage of PAS-stained area per glomerular crosssectional area. Tubulointerstitial area was assessed using
a point-counting technique (32).
Glomerulosclerosis
The degree of sclerosis in each glomerulus was graded on
a scale of 0–4 (33).
Histological Assessment of Liver
Livers were stained with hematoxylin and eosin and scored
blinded on a scale of 0–5 to determine hepatocellular cytoplasmic rarification and periportal inflammation (24).
Five to eight livers were examined per group. Ten sections
were scored and averaged to obtain an overall score for
each liver.
Immunohistochemistry
Four-micrometer aorta paraffin sections were stained for
nitrotyrosine, 4-hydroxynonenal (4-HNE), and vascular
cell adhesion molecule-1 (VCAM-1). Four-micrometer kidney sections were stained for collagen IV and nitrotyrosine
(27,28).
Sections were examined under light microscopy
(Olympus BX-50; Olympus Optical) and digitized with a
high-resolution camera. Digital quantifications (Image Pro
Plus v6.0; Media Cybernetics, Bethesda, MD) were performed in a blinded manner.
Metabolic Parameters
dh404 had no effect on systolic blood pressure in diabetic
mice (control mice 129 6 2 vs. diabetic mice 129 6 1,
130 6 1, and 134 6 1 mmHg for 3, 10, and 20 mg/kg
dh404, respectively). Body weights of STZ diabetic mice
were significantly lower than nondiabetic controls (Table 1). However, diabetic mice treated with lower doses
of dh404 (3 and 10 mg/kg/day) exhibited higher body
weights compared with both vehicle-treated diabetic
mice and diabetic mice treated with the highest dose
of 20 mg/kg dh404. Vehicle-treated diabetic mice also
displayed significantly higher kidney–, liver–, and lung–
to–body weight ratios compared with nondiabetic mice.
Treatment of mice with 3 and 10 mg/kg/day dh404 attenuated the increase in kidney–to–body weight ratio associated with diabetes (Table 1). Heart–to–body weight
ratios were unaffected by diabetes and dh404 treatment. All diabetic mice displayed increased food and
water intake with significantly increased urine output
regardless of treatment (Table 2). Diabetic mice had
significant increases in HbA1c and blood glucose levels
compared with nondiabetic mice regardless of treatment (Table 2). Diabetes was associated with increases
in total cholesterol, triglycerides, and HDL and LDL
levels. All doses of dh404 reduced these parameters,
although not to levels seen in the control mice, with
the exception of the 20 mg/kg/day treatment, in which
triglycerides and LDLs were not affected by dh404.
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dh404 Improves End-Organ Injury in Diabetic Mice
Diabetes Volume 63, September 2014
Table 1—Basic characteristics of nondiabetic and diabetic ApoE2/2 mice treated with vehicle (SO) or dh404 at conclusion of
18-week study
ND+SO
(n = 9)
ND+dh-20
(n = 8)
D+SO
(n = 5)
D+dh-3
(n = 7)
D+dh-10
(n = 8)
BW, g
30.7 6 0.9
Right kidney weight/BW, mg/g
6.8 6 0.2
D+dh-20
(n = 9)
31.1 6 0.6
21.8 6 0.4***
27.5 6 0.7**,###
26.8 6 0.6 ***,###
23.0 6 0.6***
6.4 6 0.3
11.1 6 0.4***
8.4 6 0.2*,##
8.4 6 0.7**,##
9.0 6 0.6**,##
Left kidney weight/BW, mg/g
6.9 6 0.1
6.9 6 0.3
10.5 6 0.3***
8.4 6 0.1#
7.7 6 0.5###
9.3 6 0.7***
Liver weight/BW, mg/g
42.5 6 1.5
43.9 6 1.2
59.7 6 1.3**
52.1 6 4.2*
59.6 6 2.0***
58.8 6 3.7***
Lung weight/BW, mg/g
5.8 6 0.2
5.9 6 0.2
7.5 6 0.4***
6.6 6 0.2
6.6 6 0.2
7.3 6 0.2***
Heart weight/BW, mg/g
4.9 6 0.1
5.6 6 0.6
5.9 6 0.2
5.5 6 0.3
4.8 6 0.1
6.0 6 0.4
Data are presented as mean 6 SEM. BW, body weight; D, diabetic; dh-3, -10, and -20, dh404 at 3, 10, and 20 mg/kg/day; ND,
nondiabetic. *P , 0.05, **P , 0.01, ***P , 0.001 vs. ND+SO; #P , 0.05, ##P , 0.01, ###P , 0.001 vs. D+SO.
Atherosclerotic Lesions
En Face
Diabetes induced a significant increase in total plaque in
ApoE2/2 mice, and this increase was observed in the arch,
thoracic, and abdominal regions of the aorta. Treatment
of diabetic mice with dh404 at 3 and 10 mg/kg/day significantly reduced plaque within these regions. However,
treatment with 20 mg/kg/day dh404 failed to reduce atherosclerotic plaque (Fig. 1A–E).
Aortic Sinus Lesions
Diabetes significantly increased lesion deposition within
the aortic sinus after 20 weeks of diabetes (Fig. 1F and G).
This increase was significantly attenuated after 18 weeks
of dh404 treatment at 3 and 10 mg/kg/day. Treatment
with 20 mg/kg/day dh404 failed to reduce diabetesassociated lesions.
Adhesion and Inflammatory Gene and Protein
Expression in Aorta
Gene expression of the inflammatory mediators tumor
necrosis factor-a (TNF-a) and monocyte chemotactic
protein-1 (MCP-1) was significantly upregulated in diabetic aorta. This was significantly attenuated after 5
weeks of dh404 treatment at 10 mg/kg/day but not at
20 mg/kg/day (Fig. 2A and B). No significant differences
were observed in CD36, intracellular adhesion molecule-1
(ICAM-1), p65, and VCAM-1 gene expression between
vehicle-treated diabetic and nondiabetic mice (Fig. 2C–F).
However, 10 mg/kg/day of dh404 reduced the expression
of ICAM-1 and VCAM-1 when compared with vehicletreated counterparts. VCAM-1 protein levels were significantly increased in diabetic plaque (P , 0.05) and showed
a trend toward a reduction after 10 and 20 mg/kg/day
dh404 treatment (Fig. 2G and H).
Oxidative Stress in Aortas
Analysis of 4-HNE, a marker of lipid hydroperoxides,
within the vessel walls (Supplementary Fig. 3) and plaques
(Supplementary Fig. 4) revealed a trend toward an increase in diabetic mice. This was reduced after treatment
with 3 mg/kg/day dh404 in the vessel wall, although this
did not reach statistical significance. At the highest dose
of dh404, this trend was lost in vessel walls of the diabetic
Table 2—Metabolic parameters of nondiabetic and diabetic ApoE2/2 mice treated with vehicle (SO) or dh404 at conclusion of
18-week study
ND+SO
(n = 9)
ND+dh-20
(n = 8)
D+SO
(n = 5)
D+dh-3
(n = 7)
D+dh-10
(n = 8)
D+dh-20
(n = 9)
HbA1c, %
4.3 6 0.1
3.5 6 0.1
13.2 6 1.0
10.4 6 1.9
10.6 6 1.0
10.5 6 1.4
HbA1c, mol/mol
23 6 1.1
15 6 1.1
121 6 10.9***
90 6 20.8***
92 6 10.9***
91 6 15.3***
Blood glucose, mmol/L
8.9 6 0.7
8.2 6 0.4
22.8 6 2.0***
20.7 6 2.9***
23.9 6 1.6***
21.7 6 2.6***
Total cholesterol, mmol/L
10.5 6 0.6
14.0 6 1.4
21.9 6 1.3***
14.4 6 1.5##
15.2 6 1.4##
17.6 6 1.6**,#
Triglycerides, mmol/L
1.9 6 0.4
1.3 6 0.3
7.3 6 1.0***
4.4 6 0.8*,#
4.0 6 0.4*,#
5.3 6 0.9**
HDL, mmol/L
2.2 6 0.1
3.1 6 0.3*
4.3 6 0.3***
2.7 6 0.3##
3.1 6 0.3*,#
3.2 6 0.3*,#
LDL, mmol/L
7.5 6 0.4
10.4 6 1.0
14.4 6 0.9***
9.8 6 1.1#
10.3 6 1.1
12.0 6 1.1**
Plasma AST, units/L
155 6 34
103 6 17
201 6 82
153 6 42
233 6 74
187 6 72
Plasma ALT, units/L
Water intake, mL/24 h
43 6 8
29 6 16
56 6 17
50 6 20
100 6 39
51 6 33
4.1 6 0.4
0.7 6 0.2
24.0 6 1.6***
17.1 6 3.0***
19.4 6 2.2***
16.6 6 3.9***
Food intake, g/24 h
2.4 6 0.2
3.2 6 0.2
5.2 6 0.4***
3.8 6 0.3*
4.4 6 0.3***
4.9 6 0.5***
Urinary output, mL/24 h
1.0 6 0.2
1.9 6 0.2
22.5 6 2.0***
13.6 6 2.7**
14.7 6 2.3**
16.7 6 4.1***
Data are presented as mean 6 SEM. D, diabetic; dh-3, -10, and -20, dh404 at 3, 10, and 20 mg/kg/day; ND, nondiabetic. *P , 0.05,
**P , 0.01, ***P , 0.001 vs. ND+SO; #P , 0.05, ##P , 0.01 vs. D+SO.
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Figure 1—Diabetes-associated lesion formation in the aorta and aortic sinus is attenuated by dh404 after 18 weeks of treatment. Aortas
were stained with Sudan IV Herxheimer’s solution, and plaques were stained red (A). Treatment with dh404 at 3 and 10 mg/kg/day resulted
in an attenuation of total plaque formation in the aortas of diabetic mice (B), and similar effects were found in the aortic arch (C), thoracic (D),
and abdominal regions (E). F: Cryosections of aortic sinus were stained with Oil Red O to detect plaque formation after 18 weeks of dh404
treatment. G: At 3 and 10 mg/kg/day, plaque formation was significantly attenuated in diabetic mice when compared with their vehicletreated counterparts. Data are mean 6 SEM. **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05, ##P < 0.01 vs. D+SO; ^P < 0.05, ^^P < 0.01
vs. as indicated. Abd, abdominal region; Arch, aortic arch; D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day;
ND, nondiabetic mice; Thor, thoracic.
cohort. Plaque data showed a trend toward a reduction
in 4-HNE staining in diabetic mice treated with 10 and
20 mg/kg/day dh404.
Analysis of nitrotyrosine, a marker of protein oxidative/
nitrosative stress, within plaque (Supplementary Fig. 5)
revealed a trend toward an increase in the diabetic cohort, as observed previously by us in diabetic mice (27).
This trend was lessened after treatment with dh404,
with 20 mg/kg/day showing the greatest reduction in
nitrotyrosine staining, albeit that this did not reach statistical significance.
Oxidative Stress in Urine and Plasma
Urinary 8-isoprostane, 8-OHdG, and plasma dROMs were
all significantly upregulated in vehicle-treated diabetic
mice. dh404 significantly attenuated these measures at
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Figure 2—Adhesion and inflammatory markers in aorta are reduced by dh404. The gene expression of adhesion and inflammatory markers
in the aorta was assessed by qRT-PCR after 5 weeks of treatment (A–F). VCAM-1 protein expression was examined using immunohistochemistry in aortic plaque (G), and the quantitation is shown in H. Data are mean 6 SEM. For qRT-PCR analysis, n = 6–10 (MCP-1), 7–10
(TNF-a), and 8–10 (CD36, ICAM-1, p65, and VCAM-1) aortas/group. *P < 0.05, **P < 0.01 vs. ND+SO; #P < 0.05, ##P < 0.01 vs. D+SO.
A.U., arbitrary units; D, diabetic mice dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; ND, nondiabetic mice.
all doses tested (Fig. 3A–C). Nitrotyrosine levels were
assessed in the tubulointerstitial region of the kidney,
and the diabetes-associated increase was significantly
decreased at all concentrations of dh404 (Fig. 3D and
E; P , 0.001 vs. nondiabetic control subjects).
Kidney Antioxidant and Inflammatory Gene Expression
The expression levels of Nrf2-responsive antioxidant
genes in kidney cortex, after 5 weeks of dh404 treatment,
are shown in Fig. 4A. NAD(P)H quinone oxidoreductase 1
(NQO1) was upregulated by diabetes and after treatment
with 10 and 20 mg/kg/day dh404. Similarly, expression of
glutathione-S (GSH-S) transferase was increased by diabetes but further elevated after dh404 treatment. Glutathione peroxidase (GPx) 1 expression increased in response to
dh404 treatment, reaching significance at 20 mg/kg/day
dh404 in diabetic kidneys.
Inflammatory gene expression is shown in Fig. 4B. Despite no change in interleukin-6 (IL-6) expression, MCP-1
gene expression was increased by diabetes and further increased after 10 and 20 mg/kg/day dh404, with significance
reached at the highest dose of dh404. The p65 subunit of
nuclear factor-kB (NF-kB) increased after dh404 treatment, reaching significance at 20 mg/kg/day dh404.
Renal Functional Parameters
Induction of diabetes led to a significant increase in the
urinary albumin-to-creatinine ratio, which was significantly reduced by all doses of dh404 (Fig. 4C). Elevations
in plasma cystatin C are an indication of declining glomerular filtration (30). Plasma cystatin C levels were significantly lower in vehicle-treated diabetic mice compared
with nondiabetic controls (Fig. 4D), consistent with renal
hyperfiltering (35). Treatment with dh404 did not alter
plasma cystatin C levels, implying that dh404 did not
worsen renal function at the different doses.
Kidney Morphology
Glomerular Injury
The percentage of PAS-positive material, indicative of
mesangial expansion, was significantly increased in
diabetic glomeruli (Fig. 4E and F). Furthermore, the
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Figure 3—Diabetes-associated oxidative stress is attenuated by dh404 after 18 weeks of treatment. Oxidative stress was assessed by
urine levels of 8-isoprostane (A) and 8-OHdG (B) and plasma levels of dROMs (C). All of these markers were attenuated in the diabetic mice
by dh404. Nitrotyrosine immunostaining revealed that diabetes was associated with an increase in nitrotyrosine protein expression, and
this was attenuated by dh404 at all doses (D and E). Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ND+SO; #P < 0.05,
##P < 0.01, ###P < 0.001 vs. D+SO. D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; ND, nondiabetic mice; U.Carr,
Carratelli units.
glomerulosclerosis index (GSI) of diabetic glomeruli was
significantly higher than that of nondiabetic controls
(Fig. 4G). Both mesangial expansion and GSI were attenuated in diabetic mice treated with dh404 at 3 mg/kg/day,
but this was not observed at other doses of dh404.
Tubulointerstitial Injury
Tubulointerstitial area assessment revealed a significant
increase in diabetic kidneys that was significantly attenuated at all three doses of dh404 (Fig. 4H), with the most
significant reduction observed at 3 mg/kg/day. Staining
for collagen IV showed a trend toward a reduction after
3 mg/kg dh404 when compared with diabetic kidneys
treated with vehicle only (Supplementary Fig. 6).
of hepatocellular cytoplasmic rarification, an indicator of
accumulated glycogen, showed no significant differences
after treatment with dh404, albeit that a slight increase
was observed in the nondiabetic (20 mg/kg dh404) as
well as diabetic groups receiving 3 and 10 mg/kg dh404
(Supplementary Table 1). Diabetes caused a slight albeit
nonsignificant increase in the number of infiltrating inflammatory cells. The 10-mg/kg dose of dh404 caused a statistically significant increase in infiltrating inflammatory cells.
However, this was considered mild, with an infiltrate of
,50 cells per portal vein. Thus, no significant treatmentrelated adverse liver pathology was detected after 18 weeks
of dh404 treatment (Supplementary Fig. 7).
Liver Function and Pathology
Antioxidant, Inflammatory, and Fibrotic Gene and
Protein Expression in Normal Rat Kidney Cells
Liver function was not affected by dh404, as plasma levels
of ALT and AST were not significantly different among
treatment groups (Table 2). Histopathological assessment
To complement our in vivo findings, we explored in
vitro, in normal rat kidney (NRK) cells in a dose-dependent
manner, the potential impact of dh404 on Nrf2-dependent
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Figure 4—dh404 increases antioxidant as well as inflammatory gene expression after 5 weeks of treatment, improves kidney function, and
attenuates glomerulosclerosis and tubulointerstitial injury after 18 weeks of treatment. The gene expression of antioxidants NQO1, GSH-S
transferase, and GPx1 was increased in the kidney cortex by dh404 after 5 weeks of treatment (A). However, inflammatory genes such as
MCP-1 and NF-kB were also upregulated by dh404 (B). Diabetes-associated increase in urinary albumin-to-creatinine ratio (UACR) was
reduced by dh404 (C); however, dh404 treatment had no effect on plasma cystatin C levels (D). Glomerulosclerosis was assessed by
measuring PAS-stained area per glomerulus (indicative of mesangial expansion) and also scored for GSI. Photomicrographs of representative glomeruli are shown in E. Treatment of dh404 at 3 mg/kg/day for 18 weeks significantly attenuated mesangial expansion (F) and GSI
(G). The percentage of tubulointerstitial injury was determined from PAS-stained sections by point-counting and is shown in H. Data are
mean 6 SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. ND+SO; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D+SO. A.U.,
arbitrary units; D, diabetic mice; dh-3, -10, and -20, dh404 at 3, 10, or 20 mg/kg/day; gcs, glomerular cross-sectional area; ND, nondiabetic
mice.
antioxidant genes NQO1, HO-1, and GSH-S transferase
(36). Gene expression of these antioxidants was significantly upregulated by dh404, in the presence or absence
of TGF-b, with the greatest increase noted at the highest
dose of dh404 (Fig. 5A). GPx1 gene expression was increased significantly by dh404 in the presence of TGF-b.
dh404 increased GPx2 expression significantly in the absence of TGF-b, and in the presence of TGF-b, this occurred at the highest dose of dh404. GPx3 expression was
increased significantly only at the highest dose of dh404
in the presence or absence of TGF-b (Fig. 5B). Changes in
gene expression translated into changes at the protein
level, as shown for HO-1 and GPx1 after 0.5-mmol dh404
treatment (Fig. 5B).
Despite positive reductions in IL-6 gene expression
with increasing doses of dh404 (Fig. 6A), MCP-1
expression levels increased by ;20-fold at the highest
dose of dh404. p65 gene expression was unaffected by dh404 in TGF-b–untreated cells but showed
a slight trend toward an increase at the highest dose
of dh404 compared with TGF-b–treated controls
(Fig. 6A).
Treatment with TGF-b induced a significant increase
in fibronectin and collagen I and IV gene expression (Fig.
6B). Importantly, dh404 significantly attenuated the gene
expression of these profibrotic proteins, which was mirrored by a similar protein profile for fibronectin. (Fig. 6B
and C).
diabetes.diabetesjournals.org
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Figure 5—In vitro analysis of Nrf2-responsive antioxidant genes in NRK cells after dh404 treatment. NRK cells were treated with DMSO,
dh404, TGF-b+DMSO, or TGF-b+dh404 for 72 h as detailed in the RESEARCH DESIGN AND METHODS. Gene-expression profiles of HO-1, NQO1,
GSH-S transferase, GPx1, GPx2, and GPx3 are shown in A. dh404, in most cases, caused dose-dependent increases in the gene
expression of most antioxidants investigated either in the presence or absence of TGF-b treatment. Protein levels for HO-1 and GPx1
after treatment of NRK cells with 0.5 mmol dh404 for 72 h are shown in B. Data are mean 6 SEM from five separate experiments for
quantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, data were
normalized to controls (given arbitrary value of 1) as fold change and then averaged over replicate five experiments. Lane 1, DMSO; lane 2,
dh404; lane 3, TGF-b+DMSO; lane 4, TGF-b+dh404. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. CTL+DMSO; #P < 0.05, ##P <
0.01, ###P < 0.001, ####P < 0.0001 vs. TGF-b+DMSO; ^P < 0.05 CTL+dh404 vs. TGF-b+dh404. A.U., arbitrary units; CTL, control.
DISCUSSION
In the current study, the BM derivative dh404, an Nrf2
agonist, has shown beneficial effects in attenuating
diabetes-associated atherosclerosis and nephropathy, albeit
in an inverse dose-related manner. To date, however, the
role of Nrf2 in atherosclerosis remains controversial.
Studies have reported that Nrf2 may be proatherogenic
using ApoE/Nrf2 double-knockout mice in which plaque
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dh404 Improves End-Organ Injury in Diabetic Mice
Diabetes Volume 63, September 2014
Figure 6—In vitro analysis of Nrf2-responsive anti-inflammatory and fibrosis genes in NRK cells after dh404 treatment. NRK cells were
treated with either DMSO, dh404, TGF-b+DMSO, or TGF-b+dh404 for 72 h as detailed in the RESEARCH DESIGN AND METHODS. Gene expression
profiles of IL-6, MCP-1, and the p65 subunit of NF-kB are shown in A. Gene expression of fibronectin (FN), collagen I (Col I), and collagen IV
(Col IV) are shown in B. Despite decreases in IL-6 expression with increasing doses of dh404, MCP-1 showed a significant increase in gene
expression at higher doses of dh404. Collagen I and IV as well as FN gene expression were significantly attenuated by dh404. FN protein
levels, shown in C, were significantly reduced after 0.5 mmol dh404 treatment. Data are mean 6 SEM from five separate experiments for
quantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, data
were normalized to control subjects (given arbitrary value of 1) as fold change and then averaged over replicate five experiments. *P < 0.05,
**P < 0.01, ***P < 0.001, ****P < 0.0001 vs. CTL+DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 vs. TGF-b+DMSO;
^^^P < 0.001 CTL+dh404 vs. TGF-b+dh404. Lane 1, DMSO; lane 2, dh404; lane 3, TGF-b+DMSO; lane 4, TGF-b+dh404. A.U., arbitrary
units; CTL, control.
diabetes.diabetesjournals.org
and expression of the macrophage scavenger receptor
CD36 were significantly reduced (37–39). In contrast,
the absence of Nrf2 within the myeloid lineage in LDL
receptor2/2 mice aggravated both early and late stages of
atherosclerosis (40,41). Prior to the current study, the
role of Nrf2 modulation in diabetes-associated atherosclerosis remained an unexplored area.
The results of this study show for the first time that
dh404 lessens the progression of diabetes-associated
atherosclerosis. This was observed in all regions of the
aorta but appeared to be linked to the dose, with the
lower doses of 3 and 10 mg/kg significantly lessening
the progression of diabetic lesions, while the highest dose
(20 mg/kg) failed to offer vascular protection. This was
paralleled by similar reductions in proinflammatory gene
expression (TNF-a, MCP-1, ICAM-1, and VCAM-1) at
lower doses of dh404, while the 20-mg/kg dose failed to
afford such protection. Interestingly, markers of oxidative stress such as urinary 8-isoprostane, 8-OHdG, and
plasma dROMs were significantly attenuated by all concentrations of dh404. Similarly, oxidative damage to proteins within plaque, as assessed by nitrotyrosine staining,
was reduced particularly at the highest dose of 20 mg/kg
dh404, suggesting that at this dose, there is no correlation between reductions in oxidative stress and diabetesassociated atherosclerosis.
However, at lower doses, our results suggest that
dh404 mediates its diabetes-associated antiatherogenic
effects via its anti-inflammatory and antioxidative actions.
Triterpenoids have also been shown to regulate lipid
metabolism (8,27). Thus, it is possible that the observed
reductions are due in part to the action of dh404 on
lipids since we observed reductions in LDL, triglyceride
and cholesterol after dh404 treatment, albeit that these
reductions were rather modest. Additionally, the possible minor improvement in metabolic control of diabetes
by dh404 needs to be taken into consideration. However,
these rather modest changes are unlikely to have contributed to the overall improvement in atherosclerosis.
Furthermore, the lack of an effect on atherosclerosis at
the highest dose of dh404 could represent off-target
effects of dh404 that may override any potential benefits
of improved metabolic parameters in this model.
In addition to these positive effects on the diabetic
macrovasculature, we also show significant improvements in renal function after 18 weeks of dh404 treatment. This was reflected by improvements in the urinary
albumin-to-creatinine ratio, with the lower doses of
dh404 showing the greatest inhibition. It should be noted
that the finding of a reduction in urinary albumin-tocreatinine ratio contrasts with the human data of the
BEACON trial (22), in which, despite improvements in
eGFR, the urinary albumin-to-creatinine ratio significantly
increased in the BM-treated group. A number of possibilities could explain these discordant results, such as
a species-specific effect, but more likely they relate to the
timing of treatment. Indeed, human subjects had advanced
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3101
kidney disease (stage 4) prior to exposure to BM, while in
this study, mice were treated soon after the establishment
of diabetes as a preventative strategy. One of the most
prominent effects of BM in the BEAM (20) and BEACON
(22) trials included an increase in eGFR in subjects with
renal impairment. Unfortunately, most animal models of
diabetic nephropathy, including the STZ ApoE2/2 mouse,
do not have declining GFR but rather are models of hyperfiltration (35). Indeed, in this study, analysis of plasma
cystatin C showed that diabetic mouse kidneys were hyperfiltering, a known early phenomenon of kidney disease
(30). Thus, this model is unable to specifically test the
ability of Nrf2 agents such as dh404 to increase GFR.
Our structural analyses within the diabetic kidney
showed significant reductions in tubulointerstitial injury
after treatment with dh404, with the lowest dose providing the greatest protection. Similarly, only the lowest
dose of dh404 lessened the expression of the fibrosis
marker collagen IV within this region of the kidney, albeit
that these reductions fell just short of significance. This
was also accompanied by significant reductions in oxidative stress within the tubular region of the kidney after
dh404 treatment. Additionally, we also observed significant improvements in glomerulosclerosis at the lowest
dose of dh404. Our results therefore contrast with the
study of Zoja et al. (23), in which it was reported that
dh404 failed to show beneficial effects on proteinuria and
significantly worsened tubular damage in the diabetic
Zucker fat rat. Our study also contrasts with their study
in that we did not observe any adverse effects of drug
treatment on liver structure and function and failed to
detect any renal pseudotumors. Differences in outcome
between our study and that of Zoja et al. (23) may reflect
subtle disparity in drug dosage as well as differences in
drug bioavailability between species. Our findings are,
however, more in agreement with the study by Chin
et al. (24), in which it was shown that the BM analogs
RTA 405 and dh404 were well tolerated in various rodent models of type 2 diabetes. Our study is also in
agreement with the notion that activators of Nrf2 protect against diabetic nephropathy (9).
It is of interest that in the current study, the lower
doses of dh404 appear to limit end-organ injury, whereas
this benefit is lost at higher dosages. Therefore, it is
critical to determine whether the higher dose of drug
did not work in vivo due to a lack of an increase in
antioxidant production or due to an off-target effect. To
address this issue, we analyzed several Nrf2-responsive
antioxidant genes (NQO1, GSH-S transferase, and GPx1)
in kidney cortex. Indeed, the gene expression of NQO1
and GSH-S transferase are further increased in response
to 20 mg/kg dh404. In addition, cell-culture experiments
confirmed the responsiveness of Nrf2 genes to dh404 in
a dose-dependent manner. Taken together, our results
indicate that a lack of end-organ protection by the highest
dose of dh404 cannot be explained by a lack of antioxidant responsiveness to dh404.
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dh404 Improves End-Organ Injury in Diabetic Mice
Possible off-target effects of the drug that may account
for the loss of protection at higher doses were assessed.
Indeed, proinflammatory mediators such as MCP-1 were
significantly upregulated in a dose-responsive manner,
with the highest dose of dh404 resulting in an ;20-fold
increase in MCP-1 gene expression in NRK cells. Interestingly, the highest dose of dh404 also caused a significant
increase in MCP-1 expression in the kidneys of dh404treated mice. The latter finding was also accompanied by
a significant increase in the expression of the p65 subunit
of the proinflammatory transcription factor NF-kB in the
high dose–treated diabetic kidney. Similarly, higher aortic
MCP-1 expression correlated with greater plaque. Thus,
our data suggest that dh404 might act to promote inflammation at higher doses.
Our in vitro results suggest that dh404 regulates
its antifibrotic effects in the kidney, at least partly,
through the inhibition of TGF-b activity, since both
TGF-b–stimulated fibronectin and collagen I and IV expression were inhibited by dh404 in rat kidney tubular
cells. Our results are therefore consistent with previous
studies that demonstrate Nrf2-mediated renal protection through the inhibition of TGF-b promoter activity
and its downstream pathways (9,42).
Finally, these findings need to be considered in the
clinical context. The systemic exposure of dh404 at the
lower doses in this study, corrected for the potency
difference between dh404 and BM, was similar to that
observed in both the BEAM and BEACON studies. Therefore, protection from murine end-organ injury occurs at
clinically relevant exposures, whereas this beneficial effect
was lost at higher doses. Thus, as seen in the Nrf22/2
studies (37–39), there may be a therapeutic window for
this drug class, and detailed preclinical and subsequently
clinical studies with such agents will need to be tested in
a careful dose-dependent manner.
In summary, our data have shown inverse dosedependent improvements in diabetes-associated atherosclerosis and diabetic nephropathy through the use of an
Nrf2 activator, dh404, via reductions in proinflammatory
mediators and oxidative stress in our preclinical model
of STZ-induced diabetes. The antiatherogenic function
of AIM compounds, and dh404 in particular, has not
previously been explored. Our study therefore potentially extends the pharmacotherapeutic benefits of this
class of compound to the protection against diabetesassociated atherosclerosis, a major complication affecting diabetic patients. Our study also raises the possibility
that dh404 functions to lessen both diabetic kidney
injury and diabetes-associated atherosclerosis, a highly
desirable clinical outcome since cardiovascular and renal
disease often occur concurrently (43). Finally, our findings, showing greater efficacy with respect to both cardiovascular and renal outcomes at lower dh404 doses,
highlight the importance of determining the effective
pharmacokinetic range for this class of drug. Further preclinical characterization of AIM compounds is therefore
Diabetes Volume 63, September 2014
vital before one can assess the true potential of this drug
class in the clinic.
Acknowledgments. The authors thank Katherine Ververis for excellent
technical assistance with the NRK cell studies.
Funding. S.M.T. is supported by a Juvenile Diabetes Research Foundation
International Postdoctoral Fellowship.
Duality of Interest. S.M.T., A.S., N.S., D.Y.C.Y., M.E.C., T.C.K., and J.B.d.H.
report grants and nonfinancial support from Reata Pharmaceuticals, Inc. during
the conduct of the study. C.M. and K.W.W. are employees of Reata Pharmaceuticals, Inc. and report personal fees from Reata Pharmaceuticals during the conduct of the study. No other potential conflicts of interest relevant to this article
were reported.
Author Contributions. S.M.T. and J.B.d.H. wrote the manuscript,
researched data, and analyzed results. A.S., N.S., D.Y.C.Y., and T.C.K. researched
data. C.M., K.W.W., and M.E.C. reviewed and edited the manuscript. J.B.d.H. is the
guarantor of this work and, as such, had full access to all the data in the study
and takes responsibility for the integrity of the data and the accuracy of the
data analysis.
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