, colin meyer , keith w. ward , mark e. cooper , judy b

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A derivative of Bardoxolone methyl, dh404, in an inverse dose-dependent manner,

lessens diabetes-associated atherosclerosis and improves diabetic kidney disease.

Sih Min Tan1, Arpeeta Sharma

1, Nada Stefanovic

1, Derek Y.C. Yuen

1, Tom C.

Karagiannis2, Colin Meyer

3, Keith W. Ward

3, Mark E. Cooper

1, Judy B. de Haan

1

1Oxidative Stress Laboratory, Diabetic Complications Division, Baker IDI Heart and

Diabetes Institute, Melbourne, Australia

2 Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Melbourne, Australia

3Reata Pharmaceuticals Inc. 2801 Gateway Dr, Irving, TX 75063, United States.

Running title: dh404 improves end-organ injury in diabetic mice

Corresponding author:

Dr Judy B. de Haan

Address: 75 Commercial Road, Melbourne, Victoria 3004, Australia

Fax number: +61(3) 8532 1100

Phone number: +61(3) 8532 1520

Email address: [email protected]

Word count of abstract: 200

Word count of main text: 3996

Number of figures: 6

Number of tables: 2

References: 44

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For Peer Review Only

Diabetes

Diabetes Publish Ahead of Print, published online April 16, 2014

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Abstract

Oxidative stress and inflammation are inextricably linked and play essential roles in

the initiation and progression of diabetic complications such as diabetes-associated

atherosclerosis and nephropathy. Bolstering antioxidant defences is an important

mechanism to lessen oxidative stress and inflammation. In this study, we have used a

novel analogue of the Nrf2 agonist bardoxolone methyl, dh404, to investigate its

effects on diabetic macrovascular and renal injury in STZ-induced diabetic ApoE-/-

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 pro-inflammatory mediators TNF-α, ICAM-1, VCAM-

1 and MCP-1. We demonstrate that dh404 attenuates functional (urinary

albumin/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 NRK cells showed that

dh404 significantly upregulates Nrf2-responsive genes, HO-1, NQO1 and GSH-S

transferase, with inhibition of TGFβ-mediated pro-fibrotic fibronectin, collagen I and

pro-inflammatory IL-6. Higher doses of dh404 were associated with increased

expression of pro-inflammatory mediators, MCP-1 and NF-κB. These findings

suggest that this class of compound is worthy of further study to lessen diabetic

complications but that dosage needs consideration.

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Introduction

Diabetes mellitus is a common risk factor for both chronic kidney disease and

atherosclerosis[1,2]. Despite the use of conventional therapies that include blood

pressure, glucose lowering 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 recognised to be

inextricably linked[3], approaches that limit oxidative stress are likely to translate to

reduced inflammation. Limiting oxidative stress by bolstering antioxidant defences is

a novel alternative approach to antioxidant therapy that may yield more efficacious

outcomes than standard vitamin therapy[4]. One approach that has attracted

significant attention is the augmentation of antioxidant defences via an upregulation

of the NFE2-related factor 2 (Nrf2)/Keap-1 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]. Bardoxolone

methyl and other related antioxidant inflammation modulators (AIM) 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, PPARγ and 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 hypothesised 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 hyperglycaemia 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

where this compound showed a dose-dependent increase in the estimated glomerular

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filtration rate (eGFR), initially after 56 days of treatment[19] and in a second study

(BEAM) after 24 weeks of treatment in patients with advanced chronic kidney disease

and type 2 diabetes[20]. 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 endpoints 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 emphasising that this drug class needs more extensive

investigations with respect to not only renal but also cardiovascular endpoints[22].

Potential problems with toxicity and a worsening of diabetic nephropathy were also

raised in a preclinical study examining the effects of BM analogues, 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 which showed that RTA 405 and dh404

are well tolerated and did not confirm the previous report of hepatic and renal

toxicity[24].

To further clarify the effects of Nrf2 activation on diabetic renal injury and to

investigate its potential role in lessening diabetes-associated atherosclerosis, in this

study we have treated diabetic ApoE-/- mice with the BM analogue, dh404, for 18

weeks and have assessed parameters associated with renal structural and functional

injury, as well as atherosclerosis. As rodents metabolize BM to a metabolite which

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 behaviour of BM.

Indeed, BM and dh404 exhibit very similar qualitative biological properties [25] and

the same mechanism of action, where 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, pro-inflammatory mediators and protects against diabetes-associated

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atherosclerosis. In addition, dh404 improves renal structural and functional injury

while showing no detrimental effect on liver function in this diabetic model.

Materials and Methods

Bardoxolone methyl derivative, dh404

Dh404, solubilized in sesame oil (Spectrum Chemical MFG. Corp.) at 3, 10 or

20mg/kg per body weight, was administered to mice once per day by oral gavage.

Animals

Eight-week old male C57Bl/J6 ApoE-/- mice purchased from the Animal Resource

Centre (Perth, Australia) were rendered diabetic by two intraperitoneal injections of

streptozotocin (STZ, Sigma, USA) at 100mg/kg/day on two consecutive days[26-28].

In a first cohort, 10-week old diabetic mice were gavaged with either sesame

oil (SO) or 10 or 20mg/kg dh404. Additionally, a group of non-diabetic mice received

20mg/kg dh404. Aortas and kidneys were collected for qRT-PCR after 5 weeks of

gavage. A second cohort of diabetic mice were gavaged with SO or 3, 10 or 20mg/kg

dh404 for 18 weeks. Additionally, sham-injected non-diabetic mice were gavaged

with 20mg/kg dh404. Weekly blood-glucose, body-weights and blood-pressure

measurements (using non-invasive tail-cuff plethysmography[29]) were performed.

Three days prior to termination, urine was collected in 24h metabolic cages. Mice

were killed by lethal injection of 2,2,2-tribromoethanol (Sigma, St Louis, MO, USA)

and direct puncture of the right ventricle to obtain blood. Plasma was stored at -80oC.

Plasma and urine were analysed for plasma ALT, AST and urine creatinine

(Australian Specialised 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.

Evaluation of Atherosclerotic Lesion

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, NSW,

Australia).

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Lesions within the sinus were visualised after staining with Oil Red O and

quantitated as described previously[26-28].

Kidney Function Assessment

Urinary albumin to creatinine ratio was measured using a mouse albumin ELISA kit

(Bethyl Laboratories, Montgomery, TX, USA). Plasma cystatin C was measured

using a mouse cystatin C ELISA kit[30](BioVendor, 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, USA and StressMarq

Biosciences Inc, Canada, respectively). Measures were expressed relative to urinary

creatinine. Plasma was analysed for diamicron reactive oxygen metabolites (dROMs)

particularly hydroperoxides[31] using a FRAS-4 system and expressed in Carratelli

Units[32].

Histological assessment of kidney

Mesangial and tubulointerstitial area (TIA). Kidney sections were stained with

periodic-acid Schiff (PAS). Mesangial area was quantitated using Image-Pro Plus

v6.0 (Media Cybernetics, USA) and expressed as percentage of PAS-stained area per

glomerular cross sectional (gcs) area. TIA was assessed using a point-counting

technique[33].

Glomerulosclerosis. The degree of sclerosis in each glomerulus was graded on a scale

of 0-4[34].

Histological assessment of liver

Livers were stained with haematoxylin and eosin, and scored blinded on a scale from

0-5 to determine hepatocellular cytoplasmic rarification and periportal

inflammation[19]. Five to eight livers were examined per group. Ten sections were

scored and averaged to obtain an overall score for each liver.

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Immunohistochemistry

Four-µm aorta paraffin sections were stained for nitrotyrosine, 4-hydroxynonenal (4-

HNE) and VCAM-1. Four-µm kidney sections were stained for collagen IV and

nitrotyrosine[27,28].

Sections were examined under light microscopy (Olympus BX-50; Olympus

Optical) and digitised with a high-resolution camera. Digital quantifications (Image-

Pro Plus,v6.0) were performed in a blinded manner.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

RNA from kidney and aorta was extracted using TRIzolR Reagent and cDNA

synthesised as described previously[27,28]. Gene expression was determined after

real-time quantitative RT-PCR and analysed as described previously[27,28]. Gene

expression was normalised relative to 18S ribosomal RNA. Primers and probes are

shown in Supplementary Table 1.

In vitro experiments in normal rat kidney (NRK) cells

Tubular NRK52E cells (ATCC, Rockville, USA) were maintained in Dulbecco's

modified Eagle medium containing 10% serum and 25mmol/l glucose. Cells were

serum starved and pre-treated with dh404 at 0.25µM, 0.5µM, 0.75µM or vehicle

(DMSO) for 4h before stimulation with 10ng/ml TGF-β1 for 72h[35]. Treatment at

1µM dh404 and above for 48hr resulted in significant cell death (Supplementary

Figure 2). Cells were collected for qRT-PCR or Western blot analysis[28].

Statistical Analyses

Data are expressed as mean±standard error of mean (SEM) and analysed by one-way

ANOVA with Newman-Keuls post hoc testing. Statistical analyses were performed

using GraphPad Prism version6.0 (GraphPad Software, La Jolla, CA, USA). P<0.05

was considered statistically significant.

Results

Metabolic Parameters

Dh404 had no effect on systolic blood pressure in diabetic mice (diabetic mice: 129±2

vs 129±1; 130±1; 134±1 mmHg for 3, 10, 20mg/kg dh404 respectively). Body

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weights of STZ-diabetic mice were significantly lower than non-diabetic controls

(Table 1). However, diabetic mice treated with lower doses of dh404 (3 and

10mg/kg/day) exhibited higher body-weights compared with both vehicle-treated

diabetic mice and diabetic mice treated with the highest dose of 20mg/kg dh404.

Vehicle-treated diabetic mice also displayed significantly higher kidney, liver and

lung to body-weight ratios compared with non-diabetic mice. Treatment of mice with

3 and 10mg/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 non-diabetic mice regardless of treatment (Table 2). Diabetes was associated

with increases in total cholesterol, triglycerides, 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 20mg/kg/day treatment where triglycerides and LDL were not

affected by dh404.

Atherosclerotic lesions

En face: Diabetes induced a significant increase in total plaque in ApoE-/- 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 10mg/kg/day significantly reduced

plaque within these regions. However, treatment with 20mg/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,G). This increase was significantly

attenuated after 18 weeks of dh404 treatment at 3 and 10mg/kg/day. Treatment with

20mg/kg/day dh404 failed to reduce diabetes-associated lesions.

Adhesion and Inflammatory Gene and Protein Expression in Aorta

Gene expression of the inflammatory mediators TNF-α and MCP-1 was significantly

upregulated in diabetic aorta. This was significantly attenuated after 5 weeks of dh404

treatment at 10mg/kg/day but not at 20mg/kg/day (Fig.2A,B). No significant

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differences were observed in CD36, ICAM-1, p65 and VCAM-1 gene expression

between vehicle-treated diabetic and non-diabetic mice (Fig.2C-F). However,

10mg/kg/day of dh404 reduced the expression of ICAM-1 and VCAM-1 when

compared with vehicle-treated counterparts. VCAM-1 protein levels were

significantly increased in diabetic plaque (P<0.05) and showed a trend towards a

reduction after 10 and 20mg/kg/day dh404 treatment (Fig.2G,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 towards an

increase in diabetic mice. This was reduced after treatment with 3mg/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 cohort. Plaque data

showed a trend towards a reduction in 4-HNE staining in diabetic mice treated with

10 and 20mg/kg/day dh404.

Analysis of nitrotyrosine, a marker of protein oxidative/nitrosative stress,

within plaque (Supplementary Fig.5) revealed a trend towards an increase in the

diabetic cohort, as observed previously by us in diabetic mice[27]. This trend was

lessened after treatment with dh404, with 20mg/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 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,E; P<0.001 vs non-diabetic controls).

Kidney Antioxidant and Inflammatory gene expression

The expression levels of Nrf2-responsive antioxidant genes in kidney cortex, after

5weeks of dh404 treatment, are shown in Fig.4A. NAD(P)H quinone oxidoreductase

(NQO1) was upregulated by diabetes and after treatment with 10 and 20mg/kg/day

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dh404. Similarly, expression of GSH-S transferase was increased by diabetes but

further elevated after dh404 treatment. GPx1 expression increased in response to

dh404 treatment reaching significance at 20mg/kg/day dh404 in diabetic kidneys.

Inflammatory gene expression is shown in Fig.4B. Despite no change in IL-6

expression, MCP-1 gene expression was increased by diabetes and further increased

after 10 and 20mg/kg/day dh404, with significance reached at the highest dose of

dh404. The p65 subunit of NF-κB increased after dh404 treatment, reaching

significance at 20mg/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

to non-diabetic controls (Fig.4D) consistent with renal hyper-filtering[36]. 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,F). Furthermore,

the glomerulosclerosis index (GSI) of diabetic glomeruli was significantly higher than

that of non-diabetic controls (Fig.4G). Both mesangial expansion and GSI were

attenuated in diabetic mice treated with dh404 at 3mg/kg/day but this was not

observed at other doses of dh404.

Tubulointerstitial injury: TIA assessment revealed a significant increase in diabetic

kidneys which was significantly attenuated at all 3 doses of dh404 (Fig.4H) with the

most significant reduction observed at 3mg/kg/day. Staining for collagen IV showed a

trend towards a reduction after 3mg/kg dh404 when compared to diabetic kidneys

treated with vehicle only (Supplementary Fig.6).

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Liver function and pathology

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 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 non-diabetic (20mg/kg dh404) as well as diabetic

groups receiving 3 and 10mg/kg dh404 (Supplementary Table 2). Diabetes caused a

slight albeit non-significant increase in the number of infiltrating inflammatory cells.

The 10mg/kg dose of dh404 caused a statistically significant increase in infiltrating

inflammatory cells. However, this was considered mild with an infiltrate of less than

50 cells per portal vein. Thus, no significant treatment-related adverse liver pathology

was detected after 18 weeks of dh404 treatment (Supplementary Fig.5 7).

Antioxidant, inflammatory and fibrotic Gene and Protein Expression in NRK

cells

To complement our in vivo findings, we explored in vitro, in NRK cells in a dose-

dependent manner, the potential impact of dh404 on Nrf2-dependent antioxidant

genes, NQO1, heme oxygenase-1 (HO-1) and GSH-S transferase [37]. Gene

expression of these antioxidants was significantly upregulated by dh404, in the

presence or absence of TGF-β, 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-β. Dh404 increased GPx2 expression significantly in the absence of

TGF-β, and in the presence of TGF-β, 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-β (Fig.5B). Changes in gene expression translated into

changes at the protein level as shown for HO-1 and GPx1 after 0.5µM 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-β untreated cells but

showed a slight trend towards an increase at the highest dose of dh404 compared with

TGF-β treated controls (Fig.6A).

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Treatment with TGF-β induced a significant increase in fibronectin and

collagen I and IV gene expression (Fig.6B). Importantly, dh404 significantly

attenuated the gene expression of these pro-fibrotic proteins, which was mirrored by a

similar protein profile for fibronectin. (Fig.6B,C).

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 pro-atherogenic using

ApoE/Nrf2 double knockout mice in which plaque and expression of the macrophage

scavenger receptor, CD36 were significantly reduced[38-40]. In contrast, the absence

of Nrf2 within the myeloid lineage in LDL receptor knockout (LDLR-/-) mice

aggravated both early and late stages of atherosclerosis[41,42]. 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 10mg/kg

significantly lessening the progression of diabetic lesions, while the highest dose

(20mg/kg) failed to offer vascular protection. This was paralleled by similar

reductions in pro-inflammatory gene expression (TNF-α, MCP-1, ICAM-1 and

VCAM-1) at lower doses of dh404, while the 20mg/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 20mg/kg dh404,

suggesting that at this dose there is no correlation between reductions in oxidative

stress and diabetes-associated atherosclerosis.

However, at lower doses, our results suggest that dh404 mediates its diabetes-

associated anti-atherogenic 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

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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

which 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-to-creatinine ratio contrasts with the human

data of the BEACON trial where despite improvements in eGFR, the urinary albumin-

to-creatinine ratio significantly increased in the BM-treated group.[22] A number of

possibilities could explain these discordant results such as a species-specific effect but

more likely relates to the timing of treatment. Indeed, human subjects had advanced

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 DN including the STZ ApoE knockout mouse do not have declining GFR

but rather are models of hyperfiltration[36]. 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

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glomerulosclerosis at the lowest dose of dh404. Our results therefore contrast with the

study of Zoja et al.[23], where 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. may reflect subtle disparity in drug dosage as well as differences in drug

bioavailability between species. Our findings are however, more in agreement with

Chin et al.[19] where it was shown that the BM analogues, 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 present study, the lower doses of dh404 appear to

limit end-organ injury whereas this benefit is lost at higher dosage. 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 analysed 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 20mg/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.

Possible off-target effects of the drug that may account for the loss of

protection at higher doses were assessed. Indeed, pro-inflammatory 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 dh404-treated mice. The latter finding was also

accompanied by a significant increase in the expression of the p65 subunit of the pro-

inflammatory transcription factor, NF-κB, 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.

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Our in vitro results suggest that dh404 regulates its anti-fibrotic effects in the

kidney, at least partly, through the inhibition of TGF-β activity, since both TGF-β-

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-β promoter

activity and its downstream pathways[9,43].

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 Nrf2 knockout studies[38-40], 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 dose-dependent improvements in

diabetes-associated atherosclerosis and diabetic nephropathy through the use of an

Nrf2 activator, dh404, via reductions in pro-inflammatory mediators and oxidative

stress in our pre-clinical model of STZ-induced diabetes. The anti-atherogenic

function of AIM compounds, and dh404 in particular, have not previously been

explored. Our study therefore potentially extends the pharmacotherapeutic benefits of

this class of compound to the protection against DAA, 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[44].

Finally, our findings, showing greater efficacy with respect to both cardiovascular and

renal outcomes at lower dh404 doses, highlights the importance of determining the

effective pharmacokinetic range for this class of drug. Further preclinical

characterisation of AIM compounds is therefore vital before one can assess the true

potential of this drug class in the clinic.

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Acknowledgements

S.M.T. is supported by a JDRF International Postdoctoral Fellowship. We would like

to acknowledge Katherine Ververis for her excellent technical assistance with the

NRK cell studies.

A.S., N.S., T.K and D.Y.C.Y researched data. J.B.d.H. and S.M.T. wrote the

manuscript, researched data and analysed results. C.M., K.W.W. and M.E.C

reviewed/edited the manuscript. Judy B. de Haan 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.

SMT, AS, NS, DYCY, MEC, T.K and JBdH report grants and non-financial support

from Reata Pharmaceuticals Inc. during the conduct of the study.

KWW and CM are Employees of Reata Pharmaceuticals Inc. and report personal fees

from Reata Pharmaceuticals during the conduct of the study.

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in streptozotocin-induced diabetic nephropathy. Diabetes 59: 850-860.

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Figure legends

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 10mg/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). Cryosections of aortic sinus were stained with Oil Red O

to detect plaque formation after 18 weeks of dh404 treatment (F). At 3 and 10

mg/kg/day, plaque formation was significantly attenuated in diabetic mice when

compared to their vehicle-treated counterparts (G). Data are mean ± 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. ND = non-diabetic mice; SO = sesame oil; dh-3,-10,-20 = dh404 at 3, 10 or

20mg/kg/day; D = diabetic mice.

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 ± SEM. *P<0.05, **P<0.01 vs ND+SO; #P<0.05,

##P<0.01 vs D+SO. TNF-α = tumour necrosis factor-α; MCP-1 = monocyte

chemotactic protein-1; ICAM-1 = intercellular adhesion molecule-1; VCAM-1;

vascular cell adhesion molecule-1; ND = non-diabetic mice; SO = sesame oil; dh-3,-

10,-20 = dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice. For qRT-PCR analysis,

n=6-10 (MCP-1), 7-10 (TNF-α), and 8-10 (CD36, ICAM1, p65 and VCAM-1)

aortas/group.

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 ± SEM. *P<0.05,

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**P<0.01, ***P<0.001 vs ND+SO; #P<0.05,

##P<0.01,

###P<0.001 vs D+SO. ND =

non-diabetic mice; SO = sesame oil; dh-3,-10,-20 = dh404 at 3, 10 or 20mg/kg/day; D

= diabetic mice.

Figure 4: Dh404 increases antioxidant, as well as inflammatory gene expression

after 5 weeks of treatment, and 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, were increased

in the kidney cortex by dh404 after 5 weeks of treatment (A). However, inflammatory

genes such as MCP-1 and NKκB 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 Periodic acid-Schiff (PAS)-stained

area per glomerulus (indicative of mesangial expansion) and also scored for

glomerulosclerosis index (GSI). Photomicrographs of representative glomeruli are

shown in (E). Treatment of dh404 at 3mg/kg/day for 18 weeks significantly

attenuated mesangial expansion (F) and GSI (G). The percentage tubulointerstitial

injury was determined from PAS-stained sections by point-counting and is shown in

(H). Data are mean ± 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. ND = non-diabetic mice; SO = sesame oil; dh-3,-10,-

20 = dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice.

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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 either DMSO, dh404, TGF-

β+DMSO or TGF-β+dh404 for 72 hours as detailed in the methods. Gene expression

profiles of hemeoxygenase-1 (HO1), NAD(P)H dehydrogenase (quinone 1) (NQO1),

GSH-S transferase, glutathione peroxidase (GPx)1, 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-β treatment.

Protein levels for HO-1 and GPx1 after treatment of NRK cells with 0.5µM dh404 for

72hr are shown in B. Data are mean ± SEM from 5 separate experiments for qPCR

analyses (n=5) and 3 separate experiments for Western analysis (n=3). For the qPCR

studies, data was normalized to controls (given arbitrary value =1) as fold change and

then averaged over replicate 5 experiments. *P<0.05, **P<0.01, ***P<0.001,

****P<0.0001 vs CTL+DMSO; ##

P<0.01, ###

P<0.001, ####

P<0.0001 vs TGF-

β+DMSO; ^P<0.05 CTL+dh404 vs TGF-β+dh404. CTL = control; DMSO = dimethyl

sulfoxide. Lane 1 = DMSO; lane 2 = dh404; lane 3 = TGF-β+DMSO; lane 4 = TGF-

β+dh404.

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-β+DMSO or TGF-β+dh404 for 72 hours as detailed in the

methods. Gene expression profiles of IL-6, MCP-1 and the p65 subunit of NF-κB are

shown in A. Gene expression of fibronectin (FN), collagen I and 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 was significantly attenuated by dh404. FN protein

levels, shown in C, were significantly reduced after 0.5µM dh404 treatment. Data are

mean ± SEM from 5 separate experiments for qPCR analyses (n=5) and 3 separate

experiments for Western analysis (n=3). For the qPCR studies, data was normalized

to controls (given arbitrary value =1) as fold change and then averaged over replicate

5 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs CTL+DMSO;

##P<0.01,

###P<0.001,

####P<0.0001 vs TGF-β+DMSO; ^^^P<0.001 CTL+dh404 vs

TGF-β+dh404. CTL = control; DMSO = dimethyl sulfoxide. Lane 1 = DMSO; lane 2

= dh404; lane 3 = TGF-β+DMSO; lane 4 = TGF-β+dh404.

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Table 1: Basic characteristics of non-diabetic and diabetic ApoE knockout mice

treated with vehicle (sesame oil) 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)

BW, g 30.7±0.9 31.1±0.6 21.8±0.4

***

27.5±0.7

**###

26.8±0.6

***###

23.0±0.6

***

R.Kid

weight:B

W, mg/g

6.8±0.2 6.4±0.3 11.1±0.4

***

8.4±0.2

*##

8.4±0.7

**##

9.0±0.6

**##

L.Kid

weight:B

W, mg/g

6.9±0.1 6.9±0.3 10.5±0.3

***

8.4±0.1 #

7.7±0.5 ###

9.3±0.7

***

Liver

weight:B

W, mg/g

42.5±1.5 43.9±1.2 59.7±1.3

**

52.1±4.2

*

59.6±2.0

***

58.8±3.7

***

Lung

weight:B

W, mg/g

5.8±0.2 5.9±0.2 7.5±0.4

***

6.6±0.2 6.6±0.2 7.3±0.2

***

Heart

weight:B

W, mg/g

4.9±0.1 5.6±0.6 5.9±0.2 5.5±0.3 4.8±0.1 6.0±0.4

*P<0.05, **P<0.01, ***P<0.001 vs ND+SO; #P<0.05,

##P<0.01,

###P<0.001 vs

D+SO.

BW = body weight; SBP = systolic blood pressure; R.Kid = right kidney; L.Kid = left

kidney; ND+SO = non-diabetic + sesame oil; D+SO = diabetic + sesame oil; dh-3, 10,

20 = dh404 at 3, 10 and 20mg/kg/day. Data are presented as mean ± SEM.

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Table 2: Metabolic parameters of non-diabetic and diabetic ApoE knockout mice

treated with vehicle (sesame oil) 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, %

mmol/mol

4.3±0.1

23±1.1

3.5±0.1

15±1.1

13.2±1.0

121±10.9

***

10.4±1.9

90±20.8

***

10.6±1.0

92±10.9

***

10.5±1.4

91±15.3

***

Blood

glucose,

mmol/L

8.9±0.7 8.2±0.4 22.8±2.0

***

20.7±2.9

***

23.9±1.6

***

21.7±2.6

***

Total

cholesterol,

mmol/L

10.5±0.6 14.0±1.4 21.9±1.3

***

14.4±1.5 ##

15.2±1.4 ##

17.6±1.6

**#

Triglycerides,

mmol/L

1.9±0.4 1.3±0.3 7.3±1.0

***

4.4±0.8

*#

4.0±0.4

*#

5.3±0.9

**

HDL, mmol/L 2.2±0.1 3.1±0.3

*

4.3±0.3

***

2.7±0.3 ##

3.1±0.3

*#

3.2±0.3

*#

LDL, mmol/L 7.5±0.4 10.4±1.0 14.4±0.9

***

9.8±1.1 #

10.3±1.1 12.0±1.1

**

Plasma AST,

U/L

155±34 103±17 201±82 153±42 233±74 187±72

Plasma ALT,

U/L

43±8 29±16 56±17 50±20 100±39 51±33

Water intake,

ml/24 hr

4.1±0.4 0.7±0.2 24.0±1.6

***

17.1±3.0

***

19.4±2.2

***

16.6±3.9

***

Food intake,

g/24 hr

2.4±0.2 3.2±0.2 5.2±0.4

***

3.8±0.3

*

4.4±0.3

***

4.9±0.5

***

Urinary

output, ml/24

hr

1.0±0.2 1.9±0.2 22.5±2.0

***

13.6±2.7

**

14.7±2.3

**

16.7±4.1

***

*P<0.05, **P<0.01, ***P<0.001 vs ND+SO; #P<0.05,

##P<0.01,

###P<0.001 vs

D+SO.

HbA1c = glycated haemoglobin; HDL = high-density lipoprotein; LDL = low-density

lipoprotein; AST = aspartate transaminase; ALT = alanine transaminase; ND+SO =

non-diabetic + sesame oil; D+SO = diabetic + sesame oil; dh-3, 10, 20 = dh404 at 3,

10 and 20mg/kg/day. Data are presented as mean ± SEM.

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SUPPLEMENTARY SECTION

A derivative of Bardoxolone methyl, dh404, in an inverse dose-dependent manner, lessens

diabetes-associated atherosclerosis and improves diabetic kidney disease.

Sih Min Tan1, Arpeeta Sharma

1, Nada Stefanovic

1, Derek Y.C. Yuen

1, Tom C. Karagiannis

2,

Colin Meyer3, Keith W. Ward

3, Mark E. Cooper

1, Judy B. de Haan

1

1Oxidative Stress Laboratory, Diabetic Complications Division, Baker IDI Heart and

Diabetes Institute, Melbourne, Australia 2 Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Melbourne, Australia

3Reata Pharmaceuticals Inc. 2801 Gateway Dr, Irving, TX 75063, United States.

Running title: dh404 improves end-organ injury in diabetic mice

Corresponding author:

Dr Judy B. de Haan

Address: 75 Commercial Road, Melbourne, Victoria 3004, Australia

Fax number: +61(3) 8532 1100

Phone number: +61(3) 8532 1520

Email address: [email protected]

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Figure legends

Supplementary Figure 1:

Structural representation of the parent compound Bardoxolone methyl (BM) and its

derivative, dh404, showing the –CF3 modification, an important substitution where the

fluorine on dh404 provides protection from possible rodent-specific toxic metabolites.

Previously it has been shown that agents, BM and dh404, exhibit virtually identical

qualitative properties (that is, dh404 is a reliable surrogate for bardoxolone methyl activity

[1,2]), although dh404 is ~15-fold less potent. Therefore the effects seen in these studies

with dh404 could be expected to have been observed at ~15-fold lower doses of BM, if it

could have been safely dosed to rodents. This makes the pharmacology observed even more

translationally relevant, as it would put therapeutic concentrations in the same range as that

observed in CKD patients with BM.

Supplementary Figure 2: Photomicrographs of adherent NRK cells after exposure to

increasing concentrations of dh404 for 48 and 120h.

NRK cells were treated with dh404 as shown, and allowed to grow for 48 and 120hr in the

presence of the drug. Thereafter, viable cells were assessed by trypan blue exclusion staining

and counting. Significant cell loss was observed after 48h of 1µM dh404. In subsequent

experiments a concentration range of 0 - 0.75µM dh404 for 72h was selected.

Supplementary Figure 3: Immunostaining and quantitation of 4-HNE in the aortic

vessel wall:

Aortic sections were immunostained (brown staining) with an antibody against 4-HNE to

detect lipid oxidative damage within the vessel wall. Representative photomicrographs are

shown in (A). Quantitation of 4-HNE staining is shown in (B). A trend towards an increase in

4-HNE was observed in diabetic aortas treated with sesame oil and this trend was reversed in

the presence of 3mg/kg dh404, albeit that this failed to reach significance. ND = non-diabetic

mice; SO = sesame oil; dh-3,-10,-20 = dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice.

Supplementary Figure 4: Immunostaining and quantitation of 4-HNE in aortic plaque:

Aortic sections were immunostained (brown staining) with an antibody against 4-HNE to

detect lipid oxidative damage within the plaque. Representative photomicrographs are shown

in (A). Quantitation of 4-HNE staining is shown in (B). There was a diabetes-associated

increase in 4-HNE staining in the diabetic plaque (although this failed to reach significance

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by one-way ANOVA and post hoc testing). This trend was reversed at higher doses of dh404,

albeit that this failed to reach significance. ND = non-diabetic mice; SO = sesame oil; dh-3,-

10,-20 = dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice.

Supplementary Figure 5: Immunostaining and quantitation of nitrotyrosine (NT) in

aortic plaque:

Aortic sections were immunostained (brown staining) with an antibody against NT to detect

protein oxidative damage within the plaque. Representative photomicrographs are shown in

(A). Quantitation of NT staining is shown in (B). There was a diabetes-associated increase in

NT staining in the diabetic plaque (although this failed to reach significance by one-way

ANOVA and post hoc testing). This trend was reversed at higher doses of dh404, albeit that

this failed to reach significance. ND = non-diabetic mice; SO = sesame oil; dh-3,-10,-20 =

dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice.

Supplementary Figure 6: Analysis of tubulointertitial injury

Kidney sections were subjected to immunohistochemical staining with an antibody against

collagen IV. Representative photomicrographs are shown in (A). Quantitation of collagen IV

staining is shown in (B). There was a trend towards an increase in collagen IV staining in

diabetic kidney tubules and this was reversed after 3mg/kg/day dh404 treatment for 18

weeks. 10- and 20mg/kg/day dh404 did not appear to reduce this trend. ND = non-diabetic

mice; SO = sesame oil; dh-3,-10,-20 = dh404 at 3, 10 or 20mg/kg/day; D = diabetic mice.

Supplementary Figure 7: Photomicrographs of liver sections stained with haematoxylin

and eosin:

Liver sections were stained with haematoxylin and eosin to visualise hepatocytes and

inflammatory cells enabling both the semi-quantitation of hepatocellular cytoplasmic

rarification, and the extent of inflammatory infiltration from the portal vein. (A) Non-diabetic

ApoE-/- mouse liver; (B) Non-diabetic ApoE-/- mouse liver treated with 20mg/kg dh404;

(C) Diabetic ApoE-/- mouse liver treated with SO; (D) Diabetic ApoE-/- mouse liver treated

with 3mg/kg dh404; (E) Diabetic ApoE-/- mouse liver treated with 10mg/kg dh404; (F)

Diabetic ApoE-/- mouse liver treated with 20mg/kg dh404. Arrows point to infiltrating

inflammatory cells surrounding the portal vein.

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Supplementary Figure 1

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Supplementary Table 1: Primers and probes used in quantitative RT-PCR analysis.

Gene Forward Reverse Probe

TNF-α GGCTGCCCCGACT

A

CGT

TTTCTCCTGGTATG

A

GATAGCAAATC

6- FAM

TCACCCACACCGTCAG

MCP-1

(mouse)

GTCTGTGCTGACC

C

CAAGAAG

TGGTTCCGATCCAG

GTTTTTA

6- FAM

AATGGGTCCAGACATA

C

CD36 AAGCCAGCTAGAA

AAATAGAAGCATT

AGTCTCATTTAGCC

ACAGTATAGGTACA

A

6- FAM

AGAATCTGAAGAGAC

CTTAC

ICAM-1 GGAGGTGGCGGG

AAAGTT

TCCAGCCGAGGACC

ATACAG

6-FAM

CCCTGGAACTGCACG

NFκB p65

(mouse)

TCTCACATCCGAT

TTTTGATAACC

CGAGGCAGCTCCCA

GAGTT

6-FAM

AGCTCAAGATCTGCCG

VCAM-1 CTGCTCAAGTGAT

G

GGATACCA

ATCGTCCCTTTTTGT

AGACATGAAG

6-FAM

CCAAAATCCTGTGGAG

CAG

NQO1 TTCTCTGGCCGAT

TCAGAGT

GGCTGCTTGGAGCA

AAATAG

Sybr

GSH-S

transferase

AGGCTGAGCAGG

GCTGATATT

GGGTCCAGCTCTTC

CACATG

Sybr

GPx1

(mouse)

CCCCACTGCGCTC

ATGA

GGCACACCGGAGA

CCAAA

6-FAM

CGACCCCAAGTACATC

IL-6

(mouse)

GGGAAATCGTGGA

AATGAGAAA

AAGTGCATCATCGT

TGTTCATACA

6- FAM

ATTGCCATTGCACAAC

T

HO-1 (rat) ACCCCCGAGGTCA

AGCA

TCTGGTCTTTGTGTT

CCTCTGTCA

6-FAM

CTAAGACCGCCTTCCT

GPx1 (rat) ATCCCACTGCGCT

CATGAC

TCAAAGTTCCAGGA

AATGTCGTT

6-FAM

CGACCCCAAGTACATC

GPx2 (rat) GCCAGCTACTGAG

GTCTGACAGA

GAATGGGCCAAGTT

CTTCTTGT

6-FAM

TACCTTGAACTGAATG

CACT

GPx3 (rat) TACCCTTATGACG

ACCCATTCTC

GCGCACCGGACTCC

ATAT

6-FAM

CGATCCCAAGCTCAT

IL-6 (rat) AGGGAGATCTTGG

AAATGAGAAAA

TCATCGCTGTTCAT

ACAATCAGAA

6- FAM

ATTGCCATTGCACAAC

T

MCP-1

(rat)

CACGCTTCTGGGC

CTGTT

GGCTGAGACAGCA

CGTGGAT

6-FAM

TTCACAGTTGCTGCCT

G

NFκB p65

(rat)

ACCGTGCCCCCAA

CACT

CAAGGCAGCTCCCA

GAGTTC

6-FAM

AGCTCAAGATCTGCCG

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col I (rat) TGCCGATGTCGCT

ATCCA

TCTTGCAGTGATAG

GTGATGTTCTG

6- FAM

CCTTCCTGCGCCTGA

col IV (rat) CACTATGAAAACC

GTAAAGTGCCTTA

GCAAACAGAGGCC

AACGAA

6- FAM

ATTTGCGTAACTAACA

CACC

Fn (rat) CATGGCTTTAGGC

GAACCA

CATCTACATTCGGC

AGGTATGG

6- FAM

CCCCGTCAGGCTTA

18S TGTTCACCATGAG

GCTGAGATC

TGGTTGCCTGGGAA

AATCC

6-FAM

TGCTGGCACCAGACCA

GACTTGCCCTC

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Supplementary Table 2: Histopathological assessment of hepatocellular cytoplasmic

rarification in liver sections.

Treatment

Group

Number of

livers examineda

Hepatocellular

cytoplasmic rarification

score (0-5) b

Chronic periportal

inflammation score (0-5) c

ND 8 0.61 ± 0.15 0.21 ± 0.06

ND+dh-20 8 1.10 ± 0.27 0.29 ± 0.04

D+SO 5 0.59 ± 0.13 0.46 ± 0.08

D+dh-3 7 1.26 ± 0.36 0.48 ± 0.13

D+dh-10 7 1.33 ± 0.27 0.87 ± 0.23*

D+dh-20 8 0.70 ± 0.29 0.68 ± 0.2

*P<0.05 vs ND.

ND = non-diabetic; D+SO = diabetic + sesame oil; dh-3, 10, 20 = dh404 at 3, 10 and

20mg/kg/day. Data are presented as mean ± SEM. a

For each liver, 10 sections were quantitated and averaged. b To determine hepatocellular cytoplasmic rarification, the following scale was used: 0 =

normal pathology with minor lipid inclusions (normal pathology for ApoE-/- livers);

1=minimal change; 2=mild change; 3=moderate change; 4=marked change; 5=severe change

as per Chin et al.[2] c

To determine periportal inflammation, the following scale was used: 0 = minimal

infiltration (<10 infiltrating inflammatory cells in the area surrounding the portal vein);

1=minimal change (<50 infiltrating inflammatory cells); 2=mild change (50-75 inflammatory

cells); 3=moderate change (75-100 inflammatory cells); 4=marked change (100-150

inflammatory cells); 5=severe change (>150 inflammatory cells).

REFERENCES

1. Ichikawa T, Li J, Meyer CJ, Janicki JS, Hannink M, et al. (2009) Dihydro-CDDO-

trifluoroethyl amide (dh404), a novel Nrf2 activator, suppresses oxidative stress in

cardiomyocytes. PLoS One 4: e8391.

2. Chin M, Lee CY, Chuang JC, Bumeister R, Wigley WC, et al. (2013) Bardoxolone Methyl

Analog Rta 405 and Dh404 Are Well-Tolerated and Exhibit Efficacy in Rodent

Models of Type 2 Diabetes and Obesity. Am J Physiol Renal Physiol 304: F1438-

1446.

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, colin meyer , keith w. ward , mark e. cooper , judy b

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