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 BardoxoloneMethyl, dh404, in an InverseDose-Dependent MannerLessens Diabetes-AssociatedAtherosclerosis and ImprovesDiabetic Kidney DiseaseDiabetes 2014;63:3091–3103 | DOI: 10.2337/db13-1743

Oxidative stress and inflammation are inextricably linkedand play essential roles in the initiation and progressionof diabetes complications such as diabetes-associatedatherosclerosis and nephropathy. Bolstering antioxidantdefenses is an important mechanism to lessen oxidativestress and inflammation. In this study, we have used anovel analog of the NFE2-related factor 2 (Nrf2) agonistbardoxolone methyl, dh404, to investigate its effects ondiabetic macrovascular and renal injury in streptozotocin-induced diabetic apolipoprotein E2/2 mice. We show thatdh404, at lower but not higher doses, significantly less-ens diabetes-associated atherosclerosis with reduc-tions in oxidative stress (in plasma, urine, and vasculartissue) and proinflammatory mediators tumor necrosisfactor-a, intracellular adhesion molecule-1, vascular celladhesion molecule-1, and monocyte chemotactic protein-1(MCP-1). We demonstrate that dh404 attenuates func-tional (urinary albumin-to-creatinine ratio) and structural(mesangial expansion) glomerular injury and improvesrenal tubular injury. Liver functional and structural stud-ies showed that dh404 is well tolerated. Complementaryin vitro studies in normal rat kidney cells showed thatdh404 significantly upregulates Nrf2-responsive genes,heme oxygenase-1, NAD(P)H quinone oxidoreductase 1,and glutathione-S transferase, with inhibition of trans-forming growth factor-b–mediated profibrotic fibronectin,

collagen I, and proinflammatory interleukin-6. Higherdoses of dh404 were associated with increased expres-sion of proinflammatory mediators MCP-1 and nuclearfactor-kB. These findings suggest that this class ofcompound is worthy of further study to lessen diabetescomplications but that dosage needs consideration.

Diabetes is a common risk factor for both chronic kidneydisease and atherosclerosis (1,2). Despite the use of con-ventional therapies that include blood pressure, glucose-lowering, and hyperlipidemic treatments, significantnumbers of diabetic patients suffer morbidity or mor-tality as a consequence of cardiovascular complicationsand/or progress to end-stage renal failure. Novel inter-ventions are needed to satisfy this unmet clinical need tolimit multiple diabetes-associated complications.

Evidence strongly supports a role for oxidative stressand inflammation as underlying pathogenic mechanisms ofboth diabetic renal and atherogenic complications. Withoxidative stress and inflammation now recognized to beinextricably linked (3), approaches that limit oxidativestress are likely to translate to reduced inflammation. Lim-iting oxidative stress by bolstering antioxidant defenses isa novel alternative approach to antioxidant therapy that

1Oxidative Stress Laboratory, Diabetic Complications Division, Baker IDI Heart andDiabetes Institute, Melbourne, Australia2Epigenomic Medicine, Baker IDI Heart and Diabetes Institute, Melbourne,Australia3Reata Pharmaceuticals, Inc., Irving, TX

Corresponding author: Judy B. de Haan, judy.dehaan@bakeridi.edu.au.

Received 14 November 2013 and accepted 10 April 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1743/-/DC1.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

See accompanying article, p. 2904.

Diabetes Volume 63, September 2014 3091

COMPLIC

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may yield more efficacious outcomes than standard vitamintherapy (4). One approach that has attracted significantattention is the augmentation of antioxidant defenses viaan upregulation of the NFE2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway (5–9).

Numerous small-molecule activators of the Nrf2/Keap1 pathway have been identified (10,11), includingbardoxolone methyl (BM), also known as CDDO-methyl,a potent synthetic triterpenoid inducer of the phase 2response (12,13). BM and other related antioxidant in-flammation modulators (AIMs) such as CDDO-imidazole(RTA 403) have demonstrated structural and functionalimprovements in several rodent models of renal disease(14–16), along with an increase in protective Nrf2, per-oxisome proliferator–activated receptor g, and hemeoxygenase-1 (HO-1) genes (16). Given the known nega-tive effects of oxidants and inflammation on the vascu-lature and the promising antioxidant, anti-inflammatoryand tissue-protective effects of AIM compounds (13), wehypothesized that Nrf2 agonists may additionally protectthe diabetic vasculature against accelerated atherosclero-sis. Our hypothesis is strengthened by recent data show-ing an adaptive induction of Nrf2-driven antioxidantgenes in response to hyperglycemia in human endothelialcells (17) and the use of the Nrf2 activator sulforaphaneto induce Nrf2 activation of downstream target genes inendothelial cells (18).

Interest in BM was further enhanced by two recentphase 2 clinical trials in which this compound showeda dose-dependent increase in the estimated glomerularfiltration rate (eGFR) initially after 56 days of treatment(19) and in a second study (BEAM) (20) after 24 weeks oftreatment in patients with advanced chronic kidney dis-ease and type 2 diabetes. In the latter study, the positiveeffects of drug treatment persisted at 52 weeks after re-moval of the drug at 24 weeks. However, issues of drugtoxicity and off-target effects of this drug class have beenraised, particularly after the larger phase 3 clinical trial(BEACON) with primary end points of time–to–firstoccurrence of either end-stage renal disease or cardio-vascular death (21) was terminated prematurely due tounexplained adverse cardiovascular events. Recently, theresults of BEACON have been published and suggest a po-tential effect of BM on heart failure, further emphasizingthat this drug class needs more extensive investigationswith respect to not only renal but also cardiovascular endpoints (22). Potential problems with toxicity and a wors-ening of diabetic nephropathy were also raised in a pre-clinical study examining the effects of BM analogs RTA405 and dh404 on renal outcomes in diabetic Zucker fatrats (23). The issue of toxicity was subsequently addressedin a follow-up study (24) that showed that RTA 405 anddh404 are well tolerated and did not confirm the previousreport of hepatic and renal toxicity.

To further clarify the effects of Nrf2 activation on diabeticrenal injury and investigate its potential role in lessen-ing diabetes-associated atherosclerosis, in this study, we

treated diabetic apolipoprotein E–knockout (ApoE2/2) micewith the BM analog dh404 for 18 weeks and assessed param-eters associated with renal structural and functional injury aswell as atherosclerosis. As rodents metabolize BM to a me-tabolite that itself is not toxic to higher mammals but isquite toxic to rodents, BM itself cannot be used for chronicrodent studies. Therefore, tool compounds, including dh404(also known as dh-CDDO-trifluoroethyl amide; see Supple-mentary Fig. 1), are used to probe rodent pharmacology forthis class and inform on the likely clinical behavior of BM.Indeed, BM and dh404 exhibit very similar qualitativebiological properties (25) and the same mechanism of action,in which dh404 has been shown to activate Nrf2 by prevent-ing its degradation as a result of interrupting the ability ofKeap1 to bind to Nrf2. Therefore, dh404 is an appropriatesurrogate for studying this class of compound. We now pres-ent evidence, for the first time, that dh404 lowers oxidativestress and proinflammatory mediators and protects againstdiabetes-associated atherosclerosis in a dose-dependentmanner. In addition, dh404 improves renal structural andfunctional injury while showing no detrimental effect onliver function in this diabetic model.

RESEARCH DESIGN AND METHODS

BM Derivative dh404dh404, solubilized in sesame oil (SO; Spectrum Chemical) at3, 10, or 20 mg/kg body weight, was administered to miceonce per day by oral gavage.

AnimalsEight-week-old male C57Bl/J6 ApoE2/2 mice purchasedfrom the Animal Resource Centre (Perth, Australia) wererendered diabetic by two intraperitoneal injections ofstreptozotocin (STZ; Sigma-Aldrich, St. Louis, MO) at100 mg/kg/day on 2 consecutive days (26–28).

In a first cohort, 10-week-old diabetic mice weregavaged with either SO or 10 or 20 mg/kg dh404.Additionally, a group of nondiabetic mice received20 mg/kg dh404. Aortas and kidneys were collectedfor quantitative RT-PCR (qRT-PCR) after 5 weeks ofgavage. A second cohort of diabetic mice was gavagedwith SO or 3, 10, or 20 mg/kg dh404 for 18 weeks.Additionally, sham-injected nondiabetic mice were gavagedwith 20 mg/kg dh404. Weekly blood glucose, body weight,and blood pressure measurements [using noninvasivetail-cuff plethysmography (29)] were performed. Threedays prior to termination, urine was collected in 24-hmetabolic cages. Mice were killed by lethal injection of2,2,2-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 wereanalyzed for plasma alanine transaminase (ALT), aspar-tate transaminase (AST), and urine creatinine (Austra-lian Specialized Animal Pathology Laboratory, Mulgrave,Victoria, Australia). Aortas, kidney, and livers were fixedin 10% neutral buffered formalin. Kidney cortex was snapfrozen in liquid nitrogen for RNA extraction.

3092 dh404 Improves End-Organ Injury in Diabetic Mice Diabetes Volume 63, September 2014

Evaluation of Atherosclerotic LesionEn face analysis of lesions was conducted after stainingwith Sudan IV Herxheimer’s solution (26–28). Plaque areawas calculated as the proportion of aortic intimal surfacearea occupied by red-stained plaque (Adobe Photoshopv6.0.1; Adobe Systems, New South Wales, Australia).

Lesions within the sinus were visualized after stainingwith Oil Red O and quantitated as described previously(26–28).

Kidney Function AssessmentUrinary albumin-to-creatinine ratio was measured usinga mouse albumin ELISA kit (Bethyl Laboratories, Mont-gomery, TX). Plasma cystatin C was measured using amouse cystatin C ELISA kit (30) (BioVendor LaboratoryMedicine, Brno, Czech Republic).

Oxidative Stress AssessmentUrinary 8-isoprostane and 8-hydroxy-2-deoxyguanosine(8-OHdG) were assessed using commercially available EIAkits (Cayman Chemical, Ann Arbor, MI, and StressMarqBiosciences Inc, Victoria, BC, Canada, respectively).Measures were expressed relative to urinary creatinine.Plasma was analyzed for diamicron reactive oxygenmetabolites (dROMs), particularly hydroperoxides, us-ing a Free Radical Analytical System-4 (H&D srl, Parma,Italy) and expressed in Carratelli units (31).

Histological Assessment of Kidney

Mesangial and Tubulointerstitial AreaKidney sections were stained with periodic acid-Schiff(PAS). Mesangial area was quantitated using Image-ProPlus v6.0 (Media Cybernetics, Rockville, MD) and expressedas percentage of PAS-stained area per glomerular cross-sectional area. Tubulointerstitial area was assessed usinga point-counting technique (32).

GlomerulosclerosisThe degree of sclerosis in each glomerulus was graded ona scale of 0–4 (33).

Histological Assessment of LiverLivers were stained with hematoxylin and eosin and scoredblinded on a scale of 0–5 to determine hepatocellular cy-toplasmic rarification and periportal inflammation (24).Five to eight livers were examined per group. Ten sectionswere scored and averaged to obtain an overall score foreach liver.

ImmunohistochemistryFour-micrometer aorta paraffin sections were stained fornitrotyrosine, 4-hydroxynonenal (4-HNE), and vascularcell adhesion molecule-1 (VCAM-1). Four-micrometer kid-ney sections were stained for collagen IV and nitrotyrosine(27,28).

Sections were examined under light microscopy(Olympus BX-50; Olympus Optical) and digitized with ahigh-resolution camera. Digital quantifications (Image ProPlus v6.0; Media Cybernetics, Bethesda, MD) were per-formed in a blinded manner.

qRT-PCRRNA from kidney and aorta was extracted using TRIzolReagent (Invitrogen) and cDNA synthesized as describedpreviously (27,28). Gene expression was determined afterreal-time quantitative RT-PCR and analyzed as describedpreviously (27,28). Gene expression was normalized rela-tive to 18S ribosomal RNA. Primers and probes are shownin Supplementary Table 1.

In Vitro Experiments in Normal Rat Kidney CellsTubular NRK52E cells (American Type Culture Collec-tion, Rockville, MD) were maintained in DMEM con-taining 10% serum and 25 mmol/L glucose. Cells wereserum starved and pretreated with dh404 at 0.25, 0.5,or 0.75 mmol or vehicle (DMSO) for 4 h before stimu-lation with 10 ng/mL transforming growth factor-b1(TGF-b1) for 72 h (34). Treatment at $1 mmol dh404for 48 h resulted in significant cell death (SupplementaryFig. 2). Cells were collected for qRT-PCR or Western blotanalysis (28).

Statistical AnalysesData are expressed as mean 6 SEM and analyzed byone-way ANOVA with Newman-Keuls post hoc testing.Statistical analyses were performed using GraphPadPrism version 6.0 (GraphPad, La Jolla, CA). A P value,0.05 was considered statistically significant.

RESULTS

Metabolic Parametersdh404 had no effect on systolic blood pressure in diabeticmice (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/kgdh404, respectively). Body weights of STZ diabetic micewere significantly lower than nondiabetic controls (Ta-ble 1). However, diabetic mice treated with lower dosesof dh404 (3 and 10 mg/kg/day) exhibited higher bodyweights compared with both vehicle-treated diabeticmice and diabetic mice treated with the highest doseof 20 mg/kg dh404. Vehicle-treated diabetic mice alsodisplayed 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 at-tenuated the increase in kidney–to–body weight ratio as-sociated with diabetes (Table 1). Heart–to–body weightratios were unaffected by diabetes and dh404 treat-ment. All diabetic mice displayed increased food andwater intake with significantly increased urine outputregardless of treatment (Table 2). Diabetic mice hadsignificant increases in HbA1c and blood glucose levelscompared with nondiabetic mice regardless of treat-ment (Table 2). Diabetes was associated with increasesin total cholesterol, triglycerides, and HDL and LDLlevels. All doses of dh404 reduced these parameters,although not to levels seen in the control mice, withthe exception of the 20 mg/kg/day treatment, in whichtriglycerides and LDLs were not affected by dh404.

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Atherosclerotic Lesions

En FaceDiabetes induced a significant increase in total plaque inApoE2/2 mice, and this increase was observed in the arch,thoracic, and abdominal regions of the aorta. Treatmentof diabetic mice with dh404 at 3 and 10 mg/kg/day sig-nificantly reduced plaque within these regions. However,treatment with 20 mg/kg/day dh404 failed to reduce ath-erosclerotic plaque (Fig. 1A–E).

Aortic Sinus LesionsDiabetes significantly increased lesion deposition withinthe aortic sinus after 20 weeks of diabetes (Fig. 1F and G).This increase was significantly attenuated after 18 weeksof dh404 treatment at 3 and 10 mg/kg/day. Treatmentwith 20 mg/kg/day dh404 failed to reduce diabetes-associated lesions.

Adhesion and Inflammatory Gene and ProteinExpression in AortaGene expression of the inflammatory mediators tumornecrosis factor-a (TNF-a) and monocyte chemotactic

protein-1 (MCP-1) was significantly upregulated in dia-betic aorta. This was significantly attenuated after 5weeks of dh404 treatment at 10 mg/kg/day but not at20 mg/kg/day (Fig. 2A and B). No significant differenceswere observed in CD36, intracellular adhesion molecule-1(ICAM-1), p65, and VCAM-1 gene expression betweenvehicle-treated diabetic and nondiabetic mice (Fig. 2C–F).However, 10 mg/kg/day of dh404 reduced the expressionof ICAM-1 and VCAM-1 when compared with vehicle-treated counterparts. VCAM-1 protein levels were signif-icantly increased in diabetic plaque (P , 0.05) and showeda trend toward a reduction after 10 and 20 mg/kg/daydh404 treatment (Fig. 2G and H).

Oxidative Stress in AortasAnalysis 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 in-crease in diabetic mice. This was reduced after treatmentwith 3 mg/kg/day dh404 in the vessel wall, although thisdid not reach statistical significance. At the highest doseof dh404, this trend was lost in vessel walls of the diabetic

Table 1—Basic characteristics of nondiabetic and diabetic ApoE2/2 mice treated with vehicle (SO) or dh404 at conclusion of18-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 6 0.9 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***

Right kidney weight/BW, mg/g 6.8 6 0.2 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.

Table 2—Metabolic parameters of nondiabetic and diabetic ApoE2/2 mice treated with vehicle (SO) or dh404 at conclusion of18-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 43 6 8 29 6 16 56 6 17 50 6 20 100 6 39 51 6 33

Water intake, mL/24 h 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.

3094 dh404 Improves End-Organ Injury in Diabetic Mice Diabetes Volume 63, September 2014

cohort. Plaque data showed a trend toward a reductionin 4-HNE staining in diabetic mice treated with 10 and20 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 co-hort, 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 innitrotyrosine staining, albeit that this did not reach sta-tistical significance.

Oxidative Stress in Urine and PlasmaUrinary 8-isoprostane, 8-OHdG, and plasma dROMs wereall significantly upregulated in vehicle-treated diabeticmice. dh404 significantly attenuated these measures at

Figure 1—Diabetes-associated lesion formation in the aorta and aortic sinus is attenuated by dh404 after 18 weeks of treatment. Aortaswere stained with Sudan IV Herxheimer’s solution, and plaques were stained red (A). Treatment with dh404 at 3 and 10 mg/kg/day resultedin 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 dh404treatment. G: At 3 and 10 mg/kg/day, plaque formation was significantly attenuated in diabetic mice when compared with their vehicle-treated counterparts. Data are mean6 SEM. **P< 0.01, ***P< 0.001 vs. ND+SO; #P< 0.05, ##P< 0.01 vs. D+SO; ^P< 0.05, ^^P< 0.01vs. 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.

diabetes.diabetesjournals.org Tan and Associates 3095

all doses tested (Fig. 3A–C). Nitrotyrosine levels wereassessed in the tubulointerstitial region of the kidney,and the diabetes-associated increase was significantlydecreased at all concentrations of dh404 (Fig. 3D andE; P , 0.001 vs. nondiabetic control subjects).

Kidney Antioxidant and Inflammatory Gene ExpressionThe expression levels of Nrf2-responsive antioxidantgenes 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 treatmentwith 10 and 20 mg/kg/day dh404. Similarly, expression ofglutathione-S (GSH-S) transferase was increased by diabe-tes but further elevated after dh404 treatment. Glutathi-one peroxidase (GPx) 1 expression increased in response todh404 treatment, reaching significance at 20 mg/kg/daydh404 in diabetic kidneys.

Inflammatory gene expression is shown in Fig. 4B. De-spite no change in interleukin-6 (IL-6) expression, MCP-1gene expression was increased by diabetes and further in-creased after 10 and 20 mg/kg/day dh404, with significance

reached at the highest dose of dh404. The p65 subunit ofnuclear factor-kB (NF-kB) increased after dh404 treat-ment, reaching significance at 20 mg/kg/day dh404.

Renal Functional ParametersInduction of diabetes led to a significant increase in theurinary albumin-to-creatinine ratio, which was signifi-cantly reduced by all doses of dh404 (Fig. 4C). Elevationsin plasma cystatin C are an indication of declining glo-merular filtration (30). Plasma cystatin C levels were sig-nificantly lower in vehicle-treated diabetic mice comparedwith nondiabetic controls (Fig. 4D), consistent with renalhyperfiltering (35). Treatment with dh404 did not alterplasma cystatin C levels, implying that dh404 did notworsen renal function at the different doses.

Kidney Morphology

Glomerular InjuryThe percentage of PAS-positive material, indicative ofmesangial expansion, was significantly increased indiabetic glomeruli (Fig. 4E and F). Furthermore, the

Figure 2—Adhesion and inflammatory markers in aorta are reduced by dh404. The gene expression of adhesion and inflammatory markersin the aorta was assessed by qRT-PCR after 5 weeks of treatment (A–F ). VCAM-1 protein expression was examined using immunohis-tochemistry in aortic plaque (G), and the quantitation is shown in H. Data are mean6 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.

3096 dh404 Improves End-Organ Injury in Diabetic Mice Diabetes Volume 63, September 2014

glomerulosclerosis index (GSI) of diabetic glomeruli wassignificantly higher than that of nondiabetic controls(Fig. 4G). Both mesangial expansion and GSI were atten-uated in diabetic mice treated with dh404 at 3 mg/kg/day,but this was not observed at other doses of dh404.

Tubulointerstitial InjuryTubulointerstitial area assessment revealed a significantincrease in diabetic kidneys that was significantly atten-uated at all three doses of dh404 (Fig. 4H), with the mostsignificant reduction observed at 3 mg/kg/day. Stainingfor collagen IV showed a trend toward a reduction after3 mg/kg dh404 when compared with diabetic kidneystreated with vehicle only (Supplementary Fig. 6).

Liver Function and PathologyLiver function was not affected by dh404, as plasma levelsof ALT and AST were not significantly different amongtreatment groups (Table 2). Histopathological assessment

of hepatocellular cytoplasmic rarification, an indicator ofaccumulated glycogen, showed no significant differencesafter treatment with dh404, albeit that a slight increasewas observed in the nondiabetic (20 mg/kg dh404) aswell as diabetic groups receiving 3 and 10 mg/kg dh404(Supplementary Table 1). Diabetes caused a slight albeitnonsignificant increase in the number of infiltrating inflam-matory cells. The 10-mg/kg dose of dh404 caused a statis-tically significant increase in infiltrating inflammatory cells.However, this was considered mild, with an infiltrate of,50 cells per portal vein. Thus, no significant treatment-related adverse liver pathology was detected after 18 weeksof dh404 treatment (Supplementary Fig. 7).

Antioxidant, Inflammatory, and Fibrotic Gene andProtein Expression in Normal Rat Kidney CellsTo complement our in vivo findings, we explored invitro, in normal rat kidney (NRK) cells in a dose-dependentmanner, the potential impact of dh404 on Nrf2-dependent

Figure 3—Diabetes-associated oxidative stress is attenuated by dh404 after 18 weeks of treatment. Oxidative stress was assessed byurine levels of 8-isoprostane (A) and 8-OHdG (B) and plasma levels of dROMs (C). All of these markers were attenuated in the diabetic miceby dh404. Nitrotyrosine immunostaining revealed that diabetes was associated with an increase in nitrotyrosine protein expression, andthis 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.

diabetes.diabetesjournals.org Tan and Associates 3097

antioxidant genes NQO1, HO-1, and GSH-S transferase(36). Gene expression of these antioxidants was signifi-cantly upregulated by dh404, in the presence or absenceof TGF-b, with the greatest increase noted at the highestdose of dh404 (Fig. 5A). GPx1 gene expression was in-creased significantly by dh404 in the presence of TGF-b.dh404 increased GPx2 expression significantly in the ab-sence of TGF-b, and in the presence of TGF-b, this oc-curred at the highest dose of dh404. GPx3 expression wasincreased significantly only at the highest dose of dh404in the presence or absence of TGF-b (Fig. 5B). Changes ingene expression translated into changes at the proteinlevel, as shown for HO-1 and GPx1 after 0.5-mmol dh404treatment (Fig. 5B).

Despite positive reductions in IL-6 gene expressionwith increasing doses of dh404 (Fig. 6A), MCP-1expression levels increased by ;20-fold at the highestdose of dh404. p65 gene expression was unaffect-ed by dh404 in TGF-b–untreated cells but showeda slight trend toward an increase at the highest doseof dh404 compared with TGF-b–treated controls(Fig. 6A).

Treatment with TGF-b induced a significant increasein fibronectin and collagen I and IV gene expression (Fig.6B). Importantly, dh404 significantly attenuated the geneexpression of these profibrotic proteins, which was mir-rored by a similar protein profile for fibronectin. (Fig. 6Band C).

Figure 4—dh404 increases antioxidant as well as inflammatory gene expression after 5 weeks of treatment, improves kidney function, andattenuates glomerulosclerosis and tubulointerstitial injury after 18 weeks of treatment. The gene expression of antioxidants NQO1, GSH-Stransferase, and GPx1 was increased in the kidney cortex by dh404 after 5 weeks of treatment (A). However, inflammatory genes such asMCP-1 and NF-kB were also upregulated by dh404 (B). Diabetes-associated increase in urinary albumin-to-creatinine ratio (UACR) wasreduced by dh404 (C); however, dh404 treatment had no effect on plasma cystatin C levels (D). Glomerulosclerosis was assessed bymeasuring PAS-stained area per glomerulus (indicative of mesangial expansion) and also scored for GSI. Photomicrographs of represen-tative 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 aremean 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, nondiabeticmice.

3098 dh404 Improves End-Organ Injury in Diabetic Mice Diabetes Volume 63, September 2014

DISCUSSIONIn the current study, the BM derivative dh404, an Nrf2agonist, has shown beneficial effects in attenuatingdiabetes-associated atherosclerosis and nephropathy, albeit

in an inverse dose-related manner. To date, however, therole of Nrf2 in atherosclerosis remains controversial.Studies have reported that Nrf2 may be proatherogenicusing ApoE/Nrf2 double-knockout mice in which plaque

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 geneexpression of most antioxidants investigated either in the presence or absence of TGF-b treatment. Protein levels for HO-1 and GPx1after 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 forquantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, data werenormalized 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.

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Figure 6—In vitro analysis of Nrf2-responsive anti-inflammatory and fibrosis genes in NRK cells after dh404 treatment. NRK cells weretreated with either DMSO, dh404, TGF-b+DMSO, or TGF-b+dh404 for 72 h as detailed in the RESEARCH DESIGN ANDMETHODS. Gene expressionprofiles 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 geneexpression at higher doses of dh404. Collagen I and IV as well as FN gene expression were significantly attenuated by dh404. FN proteinlevels, shown in C, were significantly reduced after 0.5 mmol dh404 treatment. Data are mean 6 SEM from five separate experiments forquantitative PCR analyses (n = 5) and three separate experiments for Western blot analysis (n = 3). For the quantitative PCR studies, datawere 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., arbitraryunits; CTL, control.

3100 dh404 Improves End-Organ Injury in Diabetic Mice Diabetes Volume 63, September 2014

and expression of the macrophage scavenger receptorCD36 were significantly reduced (37–39). In contrast,the absence of Nrf2 within the myeloid lineage in LDLreceptor2/2 mice aggravated both early and late stages ofatherosclerosis (40,41). Prior to the current study, therole of Nrf2 modulation in diabetes-associated athero-sclerosis remained an unexplored area.

The results of this study show for the first time thatdh404 lessens the progression of diabetes-associatedatherosclerosis. This was observed in all regions of theaorta but appeared to be linked to the dose, with thelower doses of 3 and 10 mg/kg significantly lesseningthe progression of diabetic lesions, while the highest dose(20 mg/kg) failed to offer vascular protection. This wasparalleled by similar reductions in proinflammatory geneexpression (TNF-a, MCP-1, ICAM-1, and VCAM-1) atlower doses of dh404, while the 20-mg/kg dose failed toafford such protection. Interestingly, markers of oxida-tive stress such as urinary 8-isoprostane, 8-OHdG, andplasma dROMs were significantly attenuated by all con-centrations of dh404. Similarly, oxidative damage to pro-teins within plaque, as assessed by nitrotyrosine staining,was reduced particularly at the highest dose of 20 mg/kgdh404, suggesting that at this dose, there is no correla-tion between reductions in oxidative stress and diabetes-associated atherosclerosis.

However, at lower doses, our results suggest thatdh404 mediates its diabetes-associated antiatherogeniceffects via its anti-inflammatory and antioxidative actions.Triterpenoids have also been shown to regulate lipidmetabolism (8,27). Thus, it is possible that the observedreductions are due in part to the action of dh404 onlipids since we observed reductions in LDL, triglycerideand cholesterol after dh404 treatment, albeit that thesereductions were rather modest. Additionally, the possi-ble minor improvement in metabolic control of diabetesby dh404 needs to be taken into consideration. However,these rather modest changes are unlikely to have con-tributed to the overall improvement in atherosclerosis.Furthermore, the lack of an effect on atherosclerosis atthe highest dose of dh404 could represent off-targeteffects of dh404 that may override any potential benefitsof improved metabolic parameters in this model.

In addition to these positive effects on the diabeticmacrovasculature, we also show significant improve-ments in renal function after 18 weeks of dh404 treat-ment. This was reflected by improvements in the urinaryalbumin-to-creatinine ratio, with the lower doses ofdh404 showing the greatest inhibition. It should be notedthat the finding of a reduction in urinary albumin-to-creatinine ratio contrasts with the human data of theBEACON trial (22), in which, despite improvements ineGFR, the urinary albumin-to-creatinine ratio significantlyincreased in the BM-treated group. A number of possibil-ities could explain these discordant results, such asa species-specific effect, but more likely they relate to thetiming of treatment. Indeed, human subjects had advanced

kidney disease (stage 4) prior to exposure to BM, while inthis study, mice were treated soon after the establishmentof diabetes as a preventative strategy. One of the mostprominent effects of BM in the BEAM (20) and BEACON(22) trials included an increase in eGFR in subjects withrenal impairment. Unfortunately, most animal models ofdiabetic nephropathy, including the STZ ApoE2/2 mouse,do not have declining GFR but rather are models of hyper-filtration (35). Indeed, in this study, analysis of plasmacystatin C showed that diabetic mouse kidneys were hyper-filtering, a known early phenomenon of kidney disease(30). Thus, this model is unable to specifically test theability of Nrf2 agents such as dh404 to increase GFR.

Our structural analyses within the diabetic kidneyshowed significant reductions in tubulointerstitial injuryafter treatment with dh404, with the lowest dose pro-viding the greatest protection. Similarly, only the lowestdose of dh404 lessened the expression of the fibrosismarker collagen IV within this region of the kidney, albeitthat these reductions fell just short of significance. Thiswas also accompanied by significant reductions in oxida-tive stress within the tubular region of the kidney afterdh404 treatment. Additionally, we also observed signifi-cant improvements in glomerulosclerosis at the lowestdose of dh404. Our results therefore contrast with thestudy of Zoja et al. (23), in which it was reported thatdh404 failed to show beneficial effects on proteinuria andsignificantly worsened tubular damage in the diabeticZucker fat rat. Our study also contrasts with their studyin that we did not observe any adverse effects of drugtreatment on liver structure and function and failed todetect any renal pseudotumors. Differences in outcomebetween our study and that of Zoja et al. (23) may reflectsubtle disparity in drug dosage as well as differences indrug bioavailability between species. Our findings are,however, more in agreement with the study by Chinet al. (24), in which it was shown that the BM analogsRTA 405 and dh404 were well tolerated in various ro-dent models of type 2 diabetes. Our study is also inagreement with the notion that activators of Nrf2 pro-tect against diabetic nephropathy (9).

It is of interest that in the current study, the lowerdoses of dh404 appear to limit end-organ injury, whereasthis benefit is lost at higher dosages. Therefore, it iscritical to determine whether the higher dose of drugdid not work in vivo due to a lack of an increase inantioxidant production or due to an off-target effect. Toaddress this issue, we analyzed several Nrf2-responsiveantioxidant genes (NQO1, GSH-S transferase, and GPx1)in kidney cortex. Indeed, the gene expression of NQO1and GSH-S transferase are further increased in responseto 20 mg/kg dh404. In addition, cell-culture experimentsconfirmed the responsiveness of Nrf2 genes to dh404 ina dose-dependent manner. Taken together, our resultsindicate that a lack of end-organ protection by the highestdose of dh404 cannot be explained by a lack of antiox-idant responsiveness to dh404.

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Possible off-target effects of the drug that may accountfor the loss of protection at higher doses were assessed.Indeed, proinflammatory mediators such as MCP-1 weresignificantly upregulated in a dose-responsive manner,with the highest dose of dh404 resulting in an ;20-foldincrease in MCP-1 gene expression in NRK cells. Interest-ingly, the highest dose of dh404 also caused a significantincrease in MCP-1 expression in the kidneys of dh404-treated mice. The latter finding was also accompanied bya significant increase in the expression of the p65 subunitof the proinflammatory transcription factor NF-kB in thehigh dose–treated diabetic kidney. Similarly, higher aorticMCP-1 expression correlated with greater plaque. Thus,our data suggest that dh404 might act to promote inflam-mation at higher doses.

Our in vitro results suggest that dh404 regulatesits antifibrotic effects in the kidney, at least partly,through the inhibition of TGF-b activity, since bothTGF-b–stimulated fibronectin and collagen I and IV ex-pression were inhibited by dh404 in rat kidney tubularcells. Our results are therefore consistent with previousstudies that demonstrate Nrf2-mediated renal protec-tion through the inhibition of TGF-b promoter activityand its downstream pathways (9,42).

Finally, these findings need to be considered in theclinical context. The systemic exposure of dh404 at thelower doses in this study, corrected for the potencydifference between dh404 and BM, was similar to thatobserved in both the BEAM and BEACON studies. There-fore, protection from murine end-organ injury occurs atclinically relevant exposures, whereas this beneficial effectwas lost at higher doses. Thus, as seen in the Nrf22/2

studies (37–39), there may be a therapeutic window forthis drug class, and detailed preclinical and subsequentlyclinical studies with such agents will need to be tested ina careful dose-dependent manner.

In summary, our data have shown inverse dose-dependent improvements in diabetes-associated athero-sclerosis and diabetic nephropathy through the use of anNrf2 activator, dh404, via reductions in proinflammatorymediators and oxidative stress in our preclinical modelof STZ-induced diabetes. The antiatherogenic functionof AIM compounds, and dh404 in particular, has notpreviously been explored. Our study therefore poten-tially extends the pharmacotherapeutic benefits of thisclass of compound to the protection against diabetes-associated atherosclerosis, a major complication affect-ing diabetic patients. Our study also raises the possibilitythat dh404 functions to lessen both diabetic kidneyinjury and diabetes-associated atherosclerosis, a highlydesirable clinical outcome since cardiovascular and renaldisease often occur concurrently (43). Finally, our find-ings, showing greater efficacy with respect to both car-diovascular and renal outcomes at lower dh404 doses,highlight the importance of determining the effectivepharmacokinetic range for this class of drug. Further pre-clinical characterization of AIM compounds is therefore

vital before one can assess the true potential of this drugclass in the clinic.

Acknowledgments. The authors thank Katherine Ververis for excellenttechnical assistance with the NRK cell studies.Funding. S.M.T. is supported by a Juvenile Diabetes Research FoundationInternational 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. duringthe conduct of the study. C.M. and K.W.W. are employees of Reata Pharmaceu-ticals, Inc. and report personal fees from Reata Pharmaceuticals during the con-duct of the study. No other potential conflicts of interest relevant to this articlewere 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. researcheddata. C.M., K.W.W., and M.E.C. reviewed and edited the manuscript. J.B.d.H. is theguarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of thedata analysis.

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