RAGE-Aptamer Blocks the Development and Progressionof Experimental Diabetic NephropathyTakanori Matsui,1 Yuichiro Higashimoto,2 Yuri Nishino,1 Nobutaka Nakamura,1 Kei Fukami,3 andSho-ichi Yamagishi1

Diabetes 2017;66:1683–1695 | https://doi.org/10.2337/db16-1281

The interaction of advanced glycation end products(AGEs) and their receptor (RAGE) plays a central rolein diabetic nephropathy. We screened DNA aptamersdirected against RAGE (RAGE-aptamers) in vitro andexamined the effects on the development and progres-sion of diabetic nephropathy in streptozotocin-induceddiabetic rats. RAGE-aptamer bound to RAGE with a Kd of5.68 nmol/L and resultantly blocked the binding of AGEsto RAGE. When diabetic rats received continuous intra-peritoneal injection of RAGE-aptamer from week 7 to11 of diabetes, the increases in renal NADPH oxidase ac-tivity, oxidative stress generation, AGE, RAGE, inflamma-tory and fibrotic gene and protein levels, macrophage andextracellular matrix accumulation, and albuminuria weresignificantly suppressed, which were associated with im-provement of podocyte damage. Two-week infusion ofRAGE-aptamer just after the induction of diabetes alsoinhibited the AGE-RAGE-oxidative stress system andMCP-1 levels in the kidneys of 8-week-old diabetic ratsand simultaneously ameliorated podocyte injury and albu-minuria. Moreover, RAGE-aptamer significantly suppressedthe AGE-induced oxidative stress generation and inflam-matory and fibrotic reactions in human cultured mesangialcells. The findings suggest that continuous infusion ofRAGE-aptamer could attenuate the development and pro-gression of experimental diabetic nephropathy by blockingthe AGE-RAGE axis.

Diabetes has been an increasing global health challenge (1).Among various complications, diabetic nephropathy is themost common cause of end-stage renal disease and often isassociated with an increased risk for cardiovascular disease,

both of which could account for the high mortality rate inpatients with diabetes (2).

Over a course of days to weeks, reducing sugars can reactnonenzymatically with the amino groups of proteins andlipids to initiate a complex series of rearrangement, de-hydration, and condensation and then to form a class ofirreversibly cross-linked adducts called advanced glycationend products (AGEs) (3,4). The formation and accumulationof AGEs in various tissues progress under diabetic condi-tions (3,4). Interaction of AGEs with the receptor for AGEs(RAGE) generates oxidative stress and stimulates inflamma-tory and fibrotic reactions, which could contribute to progres-sive alteration in renal architecture and impairment of renalfunction in diabetes (5–8). Moreover, RAGE-overexpressingdiabetic mice exhibit progressive glomerulosclerosis withrenal dysfunction, whereas homozygous RAGE knockoutdiabetic mice show reduced mesangial matrix expansionand glomerular basement membrane thickening and sclero-sis with preserved renal function (9–11). These findingssuggest that suppression of the AGE-RAGE axis in the kid-ney may be a therapeutic target for diabetic nephropathy.Indeed, inhibitors of AGE formation, as well as blockade ofthe interaction of AGEs with RAGE by an exogenously ad-ministered soluble form of RAGE, attenuate the developmentand progression of experimental diabetic nephropathy(11–14). Long-term treatment with neutralizing RAGEantibodies reduces urinary albumin excretion (UAE)and increases creatinine clearance in both type 1 and type2 diabetic mice (15,16). However, randomized, double-blind, placebo-controlled trials have revealed that AGE for-mation inhibitors, such as aminoguanidine and vitamin B6,do not significantly prevent the progression of diabetic

1Department of Pathophysiology and Therapeutics of Diabetic Vascular Compli-cations, Kurume University School of Medicine, Kurume, Japan2Department of Chemistry, Kurume University School of Medicine, Kurume,Japan3Division of Nephrology, Department of Medicine, Kurume University School ofMedicine, Kurume, Japan

Corresponding author: Sho-ichi Yamagishi, shoichi@med.kurume-u.ac.jp.

Received 21 October 2016 and accepted 24 March 2017.

© 2017 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, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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nephropathy (17,18). Therefore, to develop a novel thera-peutic strategy that specifically targets RAGE rather thanAGEs is desired.

Aptamers are short, single-stranded DNA or RNA oligo-nucleotides that are selected against various kinds of targetproteins by systematic evolution of ligands by exponentialenrichment (SELEX) (19,20). Aptamers can take a three-dimensional structure in which conformation recognizes theircognate targets with high affinity and specificity (19,20).The wide range of sequences and three-dimensional foldingpatterns of oligonucleotides allow aptamers to function likeantibodies; therefore, aptamers are termed chemical anti-bodies (19,20). Pegaptanib (Macugen) was the first U.S. Foodand Drug Administration–approved RNA aptamer directedagainst the vascular endothelial growth factor 165 isoformfor the treatment of wet-type age-related macular degener-ation (21). ARC1779 and NU172, DNA-aptamers raisedagainst the A1 domain of von Willebrand factor and exositeI of a-thrombin, currently are undergoing phase II clinicaltrials (22,23). Because aptamers are potentially used as an-tagonists and agonists of target proteins (20), we screenedhigh-affinity DNA aptamers directed against RAGE (RAGE-aptamers) by using SELEX in vitro, characterized the bind-ing affinity to and antagonistic activity for RAGE, andinvestigated whether continuous infusion of RAGE-aptamercould reverse as well as prevent the development of diabeticnephropathy in streptozotocin-induced diabetic rats. More-over, we examined the effects of RAGE-aptamer on reactiveoxygen species (ROS) generation and inflammatory and fi-brotic reactions in AGE-exposed mesangial cells.

RESEARCH DESIGN AND METHODS

MaterialsBSA, D-glyceraldehyde, streptozotocin, and bovine insulinwere purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of AGEsBSA (25 mg/mL) was incubated under sterile conditionswith 0.1 mol/L D-glyceraldehyde at 37°C for 7 days (24).Control nonglycated BSA was incubated in the same condi-tions except for the absence of D-glyceraldehyde.

Immobilizing AGE-Binding V-Domain of RAGEon Agarose BeadsHuman V-domain of RAGE (vRAGE) (residues 23–121) car-rying a C-terminal hexahistidine tag sequence (Novagen,Darmstadt, Germany) was amplified by PCR. vRAGE wascoupled to a Ni Sepharose 6 Fast Flow bead matrix (GEHealthcare, Buckinghamshire, U.K.).

Preparation and Selection of RAGE-AptamerA random combinatorial single-stranded DNA library withnormal phosphate ester backbone oligonucleotides (80-mer)was synthesized, and selection of RAGE-aptamer was per-formed by using SELEX as described previously (25,26).

ELISAvRAGE solution (1 mmol/L) was incubated with 0.5 mmol/LRAGE-aptamer or control-aptamer for 30 min. Wells precoated

with 1 mg AGEs overnight were incubated with the re-action mixtures for 1 h and then with anti-RAGE poly-clonal antibody (Santa Cruz Biotechnology, Dallas, TX).After 1 h, horseradish peroxidase–conjugated anti-rabbitIgG and 3,39,5,59-tetramethylbenzidine were added to thewells, and absorbance at 450 nm was measured.

Quartz Crystal Microbalance Binding AssayThe binding affinities of control-aptamer, RAGE-aptamer,or AGE-BSA to vRAGE were measured by using a sensitive27-MHz quartz crystal microbalance (QCM) binding assay(Initium Inc., Kanagawa, Japan) as described previously (26).

AnimalsMale 6-week-old Wistar rats (Charles River LaboratoriesJapan) were used. Rats received single intraperitonealinjection of streptozotocin (60 mg/kg body weight) in10 mmol/L citrate buffer (pH 4.5). Animals with bloodglucose levels .200 mg/dL 48 h later were considered di-abetic. One unit bovine insulin (two times per week) wassubcutaneously injected into diabetic rats to maintain life,but it was not enough to consistently reduce blood glucoselevels. Nondiabetic control rats received citrate buffer alone.

Experiment 1At 7 weeks after the injection of streptozotocin or vehicle,from 9 control and 12 diabetic rats, 4 each were transferredto metabolic cages for urinalysis. After 24 h, the rats werekilled, and blood and kidneys were obtained for NADPHoxidase activity assay, real-time RT-PCR, and biochemical,immunohistochemical, and morphological analyses. Theremaining five control and eight diabetic rats receivedcontinuous intraperitoneal infusion (2 pmol/day/g bodyweight) of either control-aptamer or RAGE-aptamer by anosmotic minipump (Durect Co., Cupertino, CA). Afteranother 4 weeks, blood and kidneys were obtained for theabove-mentioned analyses. At 7 and 11 weeks after theinjection of streptozotocin or vehicle, blood pressures weremeasured by a tail-cuff sphygmomanometer by using anautomated system with a photoelectric sensor.

Experiment 2Two days after the injection of streptozotocin or vehicle,four control and four diabetic rats were killed. Theremaining 8 control and 12 diabetic rats received contin-uous intraperitoneal infusion (0.5 pmol/day/g body weight)of either control-aptamer or RAGE-aptamer by an osmoticminipump. After 2 weeks, rats were killed, and blood andkidneys were obtained for analyses.

Albuminuria was measured with a commercially availableELISA kit (Shibayagi, Shibukawa, Japan). Serum and urinary8-hydroxy-29-deoxyguanosine (8-OHdG) levels were mea-sured with an ELISA system (Nikken Seil, Shizuoka, Japan).Other blood chemistry was analyzed with standard enzy-matic methods as described previously (27). In the prelim-inary study, we found that albuminuria began to increase at2 weeks and reached a maximum at 7 weeks after the in-duction of diabetes. Therefore, we chose this time course

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and treatment duration for experiments 1 and 2. All exper-imental procedures were conducted in accordance with theNational Institutes of Health Guide for Care and Use ofLaboratory Animals and were approved by the ethical com-mittee of Kurume University School of Medicine (Kurume,Japan).

Distribution and Kinetics of [g-32P]ATP-LabeledRAGE-AptamerMale 6-week-old Wistar rats received continuous infusion(0.5 pmol/day/g body weight) of [g-32P]ATP-labeled RAGE-aptamer by an osmotic pump and killed at 0, 1, 3, and 7 daysafter the infusion. At 3 days after stopping the infusion, bloodand organs were obtained. The half-life of RAGE-aptamer wasestimated from the time required for its renal amount after7 days of infusion to become reduced by one-half.

Immunostaining and Morphological AnalysisThe kidney sections were incubated overnight at 4°C withantibodies, and the reactions were visualized with a Histo-fine Simple Stain Rat MAX-PO (MULTI) kit (Nichirei,Tokyo, Japan) (27). Antibodies raised against 8-OHdG,nitrotyrosine (StressMarq Biosciences, Victoria, BritishColumbia, Canada), AGEs, carboxymethyllysine (CML)(TransGenic, Kobe, Japan), anti-RAGE (Santa Cruz Biotech-nology), MCP-1 (Molecular Probes, Eugene, OR), intercellu-lar adhesion molecule 1 (ICAM-1) (Abcam), vascular celladhesion molecule 1 (VCAM-1) (Santa Cruz Biotechnology),connective tissue growth factor (CTGF) (Abcam), trans-forming growth factor-b (TGF-b) (Abcam), plasminogenactivator inhibitor 1 (PAI-1) (Santa Cruz Biotechnology),type I collagen (Abcam), type III collagen (Cosmo Bio, Tokyo,Japan), type IV collagen (Abcam), fibronectin (Abcam), Mac-3(Santa Cruz Biotechnology), Wilms’ tumor-1 (WT-1) (DAKO,Santa Clara, CA), and podocin (Abcam) were used for immu-nostaining and Western blot analyses. Immunohistoreactiv-ity in five different fields in each sample was measured bycellSens version 1.14 software (Olympus, Tokyo, Japan).Three-micrometer paraffin sections were stained withMasson trichrome for light microscopic analysis as de-scribed previously (28).

RT-PCRRT-PCR was performed as described previously (26). Iden-tifications of primers for rat p22phox, NADPH oxidase 1(Nox1), NADPH oxidase 2 (Nox2, also known as gp91phox),NADPH oxidase 4 (Nox4), p47phox, p67phox, RAGE,MCP-1, ICAM-1, VCAM-1, CTGF, TGF-b, PAI-1, type Icollagen, type III collagen, type IV collagen, fibronec-tin, and 18S rRNA genes were Rn00577357_m1,Rn00586652_m1, Rn00576710_m1, Rn00585380_m1,Rn00586945_m1, Rn01759078_m1, Rn00584249_m1,Rn00580555_m1, Rn00564227_m1, Rn00563627_m1,Rn00573960_g1, Rn00572010_m1, Rn01481341_m1,Rn01463848_m1, Rn01437650_g1, Rn01482927_m1,Rn00569575_m, and Hs03003631_g1, respectively.Identifiers of primers for human RAGE, MCP-1, ICAM-1,VCAM-1, CTGF, type III collagen, fibronectin, and 18S genes

were Hs00542592_g1, Hs00234140_m1, Hs99999152_m1,Hs01003372_m1, Hs00170014_m1, Hs00943809_m1,Hs01549976_m1, and Hs03003631_g1, respectively.

Measurement of NADPH Oxidase ActivityRenal NADPH oxidase activity was measured by a lumines-cence assay as described previously (29).

Cell ExperimentsHuman mesangial cells were maintained in basal mediumsupplemented with 5% FBS (Clonetics, San Diego, CA).Mesangial cells were treated with 50 mg/mL AGE-BSA ornonglycated BSA in the presence of 100 nmol/L RAGE-aptamer or control-aptamer for 4 h (ROS assay) or 2 days(RT-PCR, Western blot analysis, and cell adhesion assay)(28).

Superoxide generation was measured with carboxy-H2DFFDA (Life Technologies Japan) as described previously(26). For Western blot analyses, nitrocellulose membraneswere probed with antibodies, and immune complexes werethen visualized with an enhanced chemiluminescence de-tection system (GE Healthcare). Data were normalized bythe intensity of a-tubulin–derived signals and related to thevalue of nonglycated BSA plus control-aptamer–treatedcells. For the adhesion assay, mesangial cells were incubatedwith BCECF, AM–labeled THP-1 cells, and then fluorescentintensities of the adherent THP-1 cells were measured (29).

Statistical AnalysisAll data are presented as mean 6 SD. ANOVA followed byStudent t test was performed for statistical comparisons.P , 0.05 was considered significant.

RESULTS

Isolation and Characterization of RAGE-AptamersIn VitroIn this study, 50 clones were sequenced from the pool ofselected single-stranded DNAs to obtain 10 unique se-quences. Structural analysis revealed that all the aptamershad a bulge-loop structure. Although all the clones signif-icantly inhibited the binding of AGEs to vRAGE, therepresentative ELISA data of three clones (#1, #2, and #3)are shown in Fig. 1A; compared with control-aptamer,RAGE-aptamers decreased the signal of AGE binding tovRAGE by ;30%. Because 100 nmol/L clone #1 did notexert a cytotoxic effect on human cultured endothelial cells(data not shown), we used it for the next experiments. Weconfirmed that AGE-BSA binds to vRAGE by a QCM bind-ing assay (Fig. 1B). A sensitive 27-MHz QCM also revealedthat RAGE-aptamer binds to vRAGE with a Kd of5.68 nmol/L (Fig. 1C and Table 1). As shown in Fig. 1D,control-aptamer did not bind to vRAGE, and alteration ofthe sequence of aptamer additions led to similar results.Moreover, in the presence of RAGE-aptamer, AGE-BSAwas not able to bind to vRAGE (Fig. 1E), which contraststhe case without RAGE-aptamer (Fig. 1B). The structure ofclone #1 RAGE-aptamer used here is shown in Fig. 1F.

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Distribution and Kinetics of Infused RAGE-AptamerAs shown in Fig. 1G, when RAGE-aptamer was continuouslyadministrated for up to 7 days, it was distributed mainlyin the aorta, eyes, testis, kidneys, and heart. After stoppingthe injection, RAGE-aptamer levels gradually decreased, butthe aptamer was still detected in the aorta and kidneys atday 3 after the removal of the osmotic pump (Fig. 1H).The elimination half-life of RAGE-aptamer in the kidneywas ;4.4 6 3.2 days.

Characteristics of Animals in Experimental Design 1We first examined whether RAGE-aptamer treatment couldinhibit the progression of experimental diabetic nephropa-thy. As shown in Fig. 2A, diabetic rats at 7 weeks or age-matched control rats received continuous intraperitonealinfusion of either control-aptamer or RAGE-aptamer for an-other 4 weeks. Clinical characteristics of animals are shown inTable 2. Fasting blood glucose, glycated hemoglobin (HbA1c),blood urea nitrogen, aspartate aminotransferase (AST),

Figure 1—A: ELISA for the binding of vRAGE to AGEs in the presence of control-aptamer or RAGE-aptamers. n = 6/group. **P< 0.01 comparedwith control-aptamer. B and C: Binding affinities of AGE-BSA or clone #1 RAGE-aptamer to vRAGE immobilized on a QCM surface. D: Bindingaffinities of control-aptamer and RAGE-aptamer to vRAGE. E: Binding affinity of 1 mg/mL AGE-BSA to vRAGE in the presence of RAGE-aptamer. F: Predicted secondary structure of clone #1 RAGE-aptamer. G and H: Biodistribution and time course kinetics of [g-32P]ATP-labeledclone #1 RAGE-aptamer. Six-week-old Wistar rats received continuous intraperitoneal infusion of [g-32P]ATP-labeled RAGE-aptamer for 7 days.Then blood, urine, and several organs were obtained at the indicated time periods. [g-32P]ATP-labeled RAGE-aptamer was detected andmeasured by Cherenkov counting. n = 3/group for B–E, G, and H.

Table 1—Sequences in random regions of the selected RAGE-thioaptamers

Aptamers Sequence of random region Kd (nmol/L)

Control-aptamer ttcggCctgggGgcggcCagttcGggtccAgtcgcGggag ND

#1 RAGE-aptamer ccTgATATggTgTcAccgccgccTTAgTATTggTgTcTAc 5.68 6 1.10

#2 RAGE-aptamer tcTgTTcAggTTggTAcggTggAAggTgTgATTcAcgAgg 4.44 6 0.56

#3 RAGE-aptamer tTccAcTgAgTgccgcggAcTgTTgTTgggAggTggTgTg 12.44 6 1.52

Data are mean 6 SD. Phosphorothioate nucleotides are indicated as capital letters. ND, not determined.

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alanine aminotransferase (ALT), and ratios of kidney, liver,and heart weight to body weight in 13- and 17-week-olddiabetic rats were higher than those of control rats of the

same ages, whereas body weight and heart rate in these ratswere lower than in control rats (Table 2). Four-week RAGE-aptamer infusion did not affect these parameters of

Figure 2—A: Experimental design 1.B–G: Gene expression levels of NADPH oxidase components.H: NADPH oxidase activity. I: Serum 8-OHdGlevels. J–M: Each left panel shows representative immunostainings of 8-OHdG (J), nitrotyrosine (K), AGE (L), and CML (M) in the kidneys. Eachright panel shows the quantitative data. N: RAGE mRNA levels. O: Left panel shows representative RAGE immunostainings in the kidneys. Rightpanel shows the quantitative data. †P< 0.05, ††P< 0.01 compared with 17-week-old control rats that received control-aptamer (Con + Con-apt);‡P < 0.05, ‡‡P < 0.01 compared with 17-week-old streptozotocin-induced diabetic rats that received control-aptamer (STZ + Con-apt).

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17-week-old diabetic rats except for the ratio of kidneyweight to body weight; the increased ratio of kidney weightto body weight was significantly reduced by the treatmentwith RAGE-aptamer.

Effects of RAGE-Aptamer on AGE-RAGE-OxidativeStress System in the Kidneys of Diabetic Rats inExperimental Design 1As shown in Fig. 2B–H, gene expression levels of compo-nents of NADPH oxidase, such as p22phox, Nox1, gp91phox,Nox4, p47phox, and p67phox, and its enzymatic activity wereincreased in 17-week-old diabetic rats compared with age-matched control rats, all of which except for p67phox weresignificantly inhibited by treatment with a continuous in-fusion of RAGE-aptamer. Furthermore, compared with con-trol rats of the same age, serum 8-OHdG as well as renallevels of 8-OHdG and nitrotyrosine, oxidative stressmarkers, and AGE, CML, and RAGE levels in the kidneysof 17-week-old diabetic rats were significantly increased(Fig. 2I–O). RAGE-aptamer treatment for 4 weeks alsoinhibited increases in these parameters in diabetic rats.

Effects of RAGE-Aptamer on Inflammatory and FibroticGene and Protein Levels, Macrophage Infiltration,Extracellular Matrix Protein Accumulation, PodocyteDamage in the Kidneys, and UAE of Diabetic Rats inExperimental Design 1As shown in Fig. 3A–T, renal mRNA and protein levels ofMCP-1; ICAM-1; VCAM-1; CTGF; TGF-b; PAI-1; type I, III,and IV collagen; and fibronectin were increased in 17-week-old diabetic rats, all of which were attenuated by RAGE-

aptamer treatment. Infusion of RAGE-aptamer also signif-icantly inhibited increases in macrophage infiltration intothe kidneys and in glomerular extracellular matrix (ECM)accumulation of 17-week-old diabetic rats (Fig. 3U and V).Moreover, decreased immunostaining levels of WT-1, a piv-otal transcription factor that is exclusively expressed inpodocytes, and podocin, a protein component of the filtra-tion slits of podocytes (30), were significantly restored bythe treatment with RAGE-aptamer (Fig. 3W and X).Whereas UAE levels were significantly higher in 13- and17-week-old diabetic rats compared with age-matched con-trol rats, RAGE-aptamer treatment for 4 weeks signifi-cantly reduced the UAE levels in 17-week-old diabetic rats(Fig. 3Y).

Characteristics of Animals in Experimental Design 2We further examined whether RAGE-aptamer treatmentcould block the development of experimental diabeticnephropathy. As shown in Fig. 4A, 6-week-old diabetic ratsor age-matched control rats received continuous intraperi-toneal infusion of either control-aptamer or RAGE-aptamerfor 2 weeks. Clinical characteristics of animals are shown inTable 3. Fasting blood glucose, AST, and ALT in 8-week-olddiabetic rats were higher than those of age-matched controlrats, whereas body weight, heart rate, HDL cholesterol, andcreatinine in these diabetic rats were lower than in thecontrol rats (Table 3). No significant differences of anthro-pometric and biochemical markers were found between8-week-old diabetic rats receiving control-aptamer orRAGE-aptamer.

Table 2—Clinical characteristics of rats in experimental design 1

13-week-old rats17-week-old rats

Control STZControl +

Control-aptamerSTZ +

Control-aptamer STZ + RAGE-aptamer

n 4 4 5 4 4

Body weight (g) 463 6 12 312 6 42* 532 6 62 267 6 56† 275 6 18††

Heart rate (beats/min) 365 6 8 295 6 6* 343 6 11 283 6 8† 270 6 16††

Mean blood pressure (mmHg) 98 6 2 91 6 4** 88 6 4 87 6 4 89 6 4

Fasting blood glucose (mg/dL) 132 6 16 522 6 38** 139 6 18 471 6 18†† 492 6 54††

HbA1c (%) 5.2 6 0.2 9.9 6 0.4** 4.6 6 0.2 9.5 6 0.6†† 9.4 6 0.2††

Total cholesterol (mg/dL) 60 6 8 65 6 12 65 6 9 111 6 44† 91 6 18

HDL cholesterol (mg/dL) 40 6 6 46 6 8 45 6 7 62 6 14 58 6 14

Triglycerides (mg/dL) 95 6 44 146 6 68 124 6 40 188 6 128 226 6 124

Blood urea nitrogen (mg/dL) 14.4 6 0.8 35.2 6 7.6** 16.0 6 2 36.4 6 4.6†† 39.8 6 6.8††

Creatinine (mg/dL) 0.3 6 0.0 0.2 6 0.0* 0.3 6 0.0 0.2 6 0.0† 0.2 6 0.0††

Kidney weight/body weight (%) 0.39 6 0.02 0.68 6 0.06** 0.34 6 0.02 0.85 6 0.08†† 0.73 6 0.06‡

AST (units/L) 70 6 14 135 6 446* 61 6 4 116 6 66† 101 6 34††

ALT (units/L) 15 6 4 46 6 14** 18 6 4 63 6 34† 57 6 24††

Liver weight/body weight (%) 2.86 6 0.10 4.25 6 0.20** 2.70 6 0.24 4.99 6 0.28†† 4.68 6 0.26††

Heart weight/body weight (%) 0.29 6 0.02 0.40 6 0.02** 0.30 6 0.02 0.44 6 0.04†† 0.47 6 0.26††

Data are mean 6 SD. STZ, streptozotocin. *P , 0.05, **P , 0.01 compared with 13-week-old control rats; †P , 0.05, ††P , 0.01compared with 17-week-old control rats that received control-aptamer; ‡P , 0.05 compared with STZ-induced diabetic rats that receivedcontrol-aptamer.

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Figure 3—A–T: Inflammatory and fibrotic gene and protein expression levels. K–U, W, and X: Each left panel shows representative immunos-tainings of MCP-1 (K), ICAM-1 (L), VCAM-1 (M), CTGF (N), TGF-b (O), PAI-1 (P), type I collagen (Q), type III collagen (R), type IV collagen (S),fibronectin (T), Mac-3 (U), WT-1 (W), and podocin (X) in the kidneys. Each right panel shows the quantitative data. V: Left panel shows

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Effects of RAGE-Aptamer on AGE-RAGE-OxidativeStress System and MCP-1 Levels in the Kidneys ofDiabetic Rats in Experimental Design 2As shown in Fig. 4B–I, urinary 8-OHdG excretion, nitro-tyrosine immunostaining, AGE, CML, RAGE, and MCP-1levels in the kidneys of 8-week-old diabetic rats were sig-nificantly increased compared with control rats, all of whichwere attenuated with the treatment with RAGE-aptamer.

Effects of RAGE-Aptamer on Podocyte Damage andUAE Levels of Diabetic Rats in Experimental Design 2As shown in Fig. 4J and K, RAGE-aptamer treatment for2 weeks significantly restored the decrease in immunostain-ing levels of WT-1 and podocin in diabetic kidneys. UAElevels of 8-week-old diabetic rats were significantly in-creased compared with control rats, which were attenuatedby the treatment with RAGE-aptamer (Fig. 4L).

Effects of RAGE-Aptamer on ROS Generation,RAGE, and Inflammatory and Fibrotic Reactions inAGE-Exposed Human Mesangial CellsFifty micrograms per milliliter AGE-BSA for 4 h significantlyincreased ROS generation (Fig. 5A).MCP-1 gene expression,RAGE, ICAM-1, VCAM-1, CTGF, type III collagen, and fi-bronectin mRNA and protein levels were increased at 2 daysafter the treatment with 50 mg/mL AGE-BSA, all of whichwere significantly attenuated by the treatment of 100 nmol/LRAGE-aptamer (Fig. 5B–O). RAGE-aptamer at 100 nmol/Ldid not affect these mRNA or protein levels in nonglycatedBSA-exposed mesangial cells. Because we could not detectmeasurable levels of MCP-1 by Western blot analysis, westudied the effects of RAGE-aptamer on THP-1 cell ad-hesion to mesangial cells. As shown in Fig. 5P, 50 mg/mLAGE-BSA stimulated THP-1 cell adhesion to mesangial cells,which was inhibited by 100 nmol/L RAGE-aptamer.

DISCUSSION

We show for the first time, to our knowledge, that althoughcontinuous infusion of RAGE-aptamer for 4 weeks did notimprove hyperglycemia or affect lipid parameters or bloodpressure levels, it attenuated the increases in macrophageinfiltration into the kidneys, glomerular ECM protein ac-cumulation, and UAE levels of 17-week-old streptozotocin-induced diabetic rats and improved podocyte damage,which was associated with a significant reduction in renaloxidative stress, AGE, RAGE, and inflammatory and fibroticmarker levels (Figs. 2 and 3). Compared with age-matchedcontrol rats, ratio of kidney weight to body weight, glomer-ular ECM accumulation, and UAE levels were already in-creased after 7 weeks of diabetes in association with

significant elevations of ICAM-1, VCAM-1, CTGF, type Icollagen, and fibronectin mRNA levels in the kidneys (Fig.3), suggesting the beneficial effects of RAGE-aptamer onestablished renal damage in diabetic nephropathy. Further-more, 2-week continuous administration of RAGE-aptamerjust after the induction of diabetes also significantly sup-pressed increases in renal nitrotyrosine, AGE, and RAGElevels and urinary 8-OHdG excretion in 8-week-old diabeticrats and decreased albuminuria in association with amelio-ration of podocyte damage (Fig. 4). In addition, we foundthat RAGE-aptamer significantly inhibited AGE-induced in-creases in ROS generation and upregulation of RAGEmRNA levels in mesangial cells and subsequently reducedinflammatory and fibrotic reactions. Therefore, the currentstudy suggests that RAGE-aptamer treatment could blockthe development and progression of diabetic nephropathyin type 1 diabetic rats by suppressing the AGE-RAGE axisin the kidneys.

Aptamers have less immunogenicity over antibodies forblocking diverse target proteins and can be easily selectedwith relatively low production costs. Furthermore, becauseof their small sizes, aptamers can more efficiently penetratevarious tissues, which in concert with the above-mentionedcharacteristics can improve their clinical applicability(19,20). We developed RAGE-aptamers that specificallybind to RAGE and resultantly block binding of AGEs toRAGE in vitro. Because RAGE-aptamer at 100 nmol/L didnot affect ROS generation or inflammatory and fibroticreactions in nonglycated BSA–exposed mesangial cells(Fig. 5), phosphorothioate-modified RAGE-aptamer usedin the current experiments exhibited a significant RAGE-antagonistic activity without agonist-like properties. More-over, we found that RAGE-aptamer levels were increasedin the kidneys of 6-week-old Wistar rats for at least 7 daysafter a continuous intraperitoneal infusion. The Kd ofRAGE-aptamer for RAGE was 5.68 nmol/L, and its elimina-tion half-life was 4.4 days; RAGE-aptamer was still detectedin the kidneys at 3 days after stopping the injection. Theseobservations suggest that phosphorothioate-modifiedRAGE-aptamer may be quite stable in vivo, and continuousinfusion of RAGE-aptamer with the use of a device like aninsulin pump could become a feasible therapeutic strategyfor the treatment of diabetic nephropathy.

We demonstrated that both continuous infusion of RAGE-aptamer just after the induction of diabetes and after 7 weeksof diabetes significantly decreases renal AGE, CML, andRAGE levels in diabetic rats. We have previously shown thatneutralizing antibody raised against RAGE or an antioxidantinhibits AGE-induced ROS generation, redox-sensitive

representative Masson trichrome stainings of the kidneys. Right panel shows the quantitative data of glomerular ECM protein accumulation. Y:UAE levels. *P < 0.05, **P < 0.01 compared with 13-week-old control rats (Con); †P < 0.05, ††P < 0.01 compared with 17-week-old controlrats that received control-aptamer (Con + Con-apt); ‡P < 0.05, ‡‡P < 0.01 compared with 17-week-old streptozotocin-induced diabetic ratsthat received control-aptamer (STZ + Con-apt). Cre, creatinine; RAGE-apt, RAGE-aptamer.

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Figure 4—A: Experimental design 2. B: Urinary 8-OHdG levels. C–E: Each left panel shows representative immunostainings of nitrotyrosine (C),AGE (D), and CML (E) in the kidneys. Each right panel shows the quantitative data. F andH: RAGE andMCP-1mRNA levels.G and I–K: Each leftpanel shows representative immunostainings of RAGE (G), MCP-1 (I), WT-1 (J), and podocin (K) in the kidneys. Each right panel shows thequantitative data. L: UAE levels. †P < 0.05, ††P < 0.01 compared with 8-week-old control rats that received control-aptamer (Con + Con-apt);‡P< 0.05, ‡‡P< 0.01 comparedwith 8-week-old streptozotocin-induced diabetic rats that received control-aptamer (STZ +Con-apt). Cre, creatinine.

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transcriptional factor, nuclear factor-kB activation, andupregulation of RAGE mRNA levels in mesangial cells(31), indicating that AGE-RAGE–evoked ROS generationfurther enhances RAGE gene expression in mesangial cells,which may make a vicious cycle (31). Renal CML accumu-lation and its urinary excretion levels have been suppressedin RAGE-deficient streptozotocin-induced diabetic rats (32).Moreover, DNA aptamer raised against AGEs has reducedAGE levels in the kidneys of obese and type 2 diabetic mice(28). These findings suggest a positive feedback loop be-tween RAGE-derived ROS generation and AGE accumula-tion in diabetic kidneys. RAGE-aptamer could decrease renalAGE, CML, and RAGE levels in type 1 diabetic rats by break-ing the crosstalk between the AGE-RAGE axis and ROS.

AGE-RAGE interaction not only causes inflammatoryand fibrotic reactions in mesangial cells and diabetic kidneysbut also evokes podocyte damage through NADPH oxidase–mediated ROS generation (32–39). In the current study, wefound that treatment with RAGE-aptamer suppressed upre-gulation of mRNA levels for NADPH oxidase componentsand its enzymatic activity in the kidneys of 17-week-olddiabetic rats, which was associated with decreased serum8-OHdG as well as renal 8-OHdG and nitrotyrosine levels.Urinary excretion levels of 8-OHdG were increased in8-week-old diabetic rats, which were also attenuated bytreatment with RAGE-aptamer. Therefore, the findings sug-gest that blockade of the AGE-RAGE interaction by RAGE-aptamer inhibits inflammatory and fibrotic reactions andpodocyte damage in diabetic kidneys through suppressionof ROS production, which could lead to the reduction ofalbuminuria in type 1 diabetic rats.

Epidemiological studies have supported the concept ofmetabolic memory in the development and progression ofvascular complications in diabetes (40–43). Because AGEaccumulation could reflect cumulative hyperglycemic expo-sure and contribute to inflammatory and fibrotic reactions

and podocyte damage in diabetic kidneys through the sus-tained activation of RAGE (5–8,39,44–46), the AGE-RAGEaxis is supposed to play a central role in the phenomenon ofmetabolic memory. The observation that RAGE-aptamertreatment reduced AGE accumulation and RAGE expressionlevels in the kidneys even when given after established renalinjury may support the clinical utility of RAGE-aptamer forthe treatment of diabetic nephropathy.

LimitationsWe have already shown that glyceraldehyde-modified AGEscontribute to diabetic complications in animal models andthat their circulating levels are correlated with endothelialdysfunction and inflammatory biomarkers in high-riskpatients, including those with diabetes (27,28,36,47,48).Furthermore, in glyceraldehyde-modified AGEs bound tovRAGE with a Kd of 8.60 nmol/L (Fig. 1B), the bindingaffinity was three orders of magnitude stronger than thatof CML, carboxyethyllysine, or methylglyoxal-derivedhydroimidazolone-1 (Kd of 31.4, 27.4, and 56.7 mmol/L,respectively). For this reason, we used glyceraldehyde-modified AGEs instead of CML, carboxyethyllysine, ormethylglyoxal-derived hydroimidazolone-1, which have beenshown to cause diabetic complications and react withRAGE (5,35).

We used a dose of 60 mg/kg streptozotocin to inducediabetes because 50–65 mg/kg streptozotocin-induced di-abetic rats have exhibited hemodynamic and structuralalterations in the kidneys, a considerable part of whichresembles human diabetic nephropathy (49,50). Althoughwe cannot totally exclude the possibility that this dose maybe associated with nephrotoxicity, given the pathologicalrole of AGE-RAGE axis in diabetic nephropathy (5–8), thecurrent study suggests that RAGE-aptamer ameliorates re-nal damage in streptozotocin-induced diabetic rats by block-ing the interaction of AGEs with RAGE. However, because

Table 3—Clinical characteristics of rats in experimental design 2

6-week-old rats8-week-old rats

ControlControl+

Control-aptamerControl +

RAGE-aptamerSTZ +

Control-aptamer STZ + RAGE-aptamer

n 4 4 4 6 6

Body weight (g) 168 6 8 271 6 12* 276 6 16 184 6 32† 180 6 34

Heart rate (beats/min) 372 6 20 412 6 20* 397 6 36 303 6 29†† 312 6 27††

Mean blood pressure (mmHg) 90 6 4 100 6 4 102 6 6 84 6 15 92 6 15

Fasting blood glucose (mg/dL) 93 6 12 74 6 16 78 6 10 184 6 110† 211 6 149††

Total cholesterol (mg/dL) 71 6 8 80 6 20 76 6 8 61 6 25 52 6 10†

HDL cholesterol (mg/dL) 57 6 6 58 6 16 56 6 4 41 6 15† 38 6 7†

Triglycerides (mg/dL) 42 6 22 73 6 38 74 6 10 51 6 25 42 6 15†

Creatinine (mg/dL) 0.14 6 0.0 0.21 6 0.0** 0.28 6 0.2 0.16 6 0.0† 0.18 6 0.0

AST (units/L) 79 6 10 71 6 6 63 6 10 321 6 245† 276 6 225†

ALT (units/L) 39 6 6 21 6 2* 19 6 2 238 6 254† 112 6 86†

Data are mean6 SD. STZ, streptozotocin. *P, 0.05, **P, 0.01 between 6-week-old control rats and 8-week-old control rats that receivedcontrol-aptamer; †P , 0.05, ††P , 0.01 compared with 8-week-old control rats that received control-aptamer.

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streptozotocin-induced diabetic nephropathy is not com-pletely the same as human diabetic nephropathy, furtherstudy is needed to clarify whether RAGE-aptamer may be atherapeutic strategy for the treatment of diabetic nephrop-athy in humans.

Because RAGE-aptamer treatment for 2 weeks did notaffect the AGE-RAGE-oxidative stress system or renal injuryin nondiabetic control rats at 6 weeks old (Fig. 4), we omitteda group of RAGE-aptamer–treated controls in experiment 1.Therefore, whether RAGE-aptamer treatment for 4 weekshas an effect on 13-week-old control rats remains unclear.

We found that RAGE-aptamer treatment significantlyreduces 8-OHdG and nitrotyrosine levels in the kidneys of8- and 17-week-old diabetic rats (Figs. 2 and 4). Although

the effects of RAGE-aptamer on these oxidative stress markerswere modest, the data of mRNA expression of Nox subunits,NADPH oxidase activity, and mesangial cell experiments sup-port that ROS plays a major role in the aptamer actions. Theeffects of RAGE-aptamer on diabetic nephropathy in NADPHoxidase component knockout mice, including gp91phox- orNox1-deficient mice, would be interesting to investigate.

Funding. This work was supported in part by Grants-in-Aid for ScientificResearch C from the Ministry of Education, Culture, Sports, Science, and Technologyof Japan (grant number 16K07101 to T.M.) and by The Mitsubishi Foundation (grantnumber 20162714 to S.-i.Y.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.

Figure 5—Effect of RAGE-aptamer on ROS generation (A), and RAGE (B and J), MCP-1 (C), ICAM-1 (D and K), VCAM-1 (E and L), CTGF (F andM), type III collagen (G and N), and fibronectin (H and O) levels in and THP-1 (P) adhesion to AGE-exposed mesangial cells. Representativebands of Western blot analyses also are shown (I). Mesangial cells were treated with 50 mg/mL AGE-BSA or nonglycated BSA for 4 h (A) and2 days (B–P) in the presence of 100 nmol/L RAGE-aptamer (RAGE-apt). Total RNAs were transcribed and amplified by real-time RT-PCR (B–H).Data were normalized by the intensity of 18S rRNA–derived signals and then related to the value obtained with nonglycated BSA plus control-aptamer (Con-apt)–treated cells. For Western blot analyses (I–O), 10 mg of proteins were extracted from mesangial cells and then separated bySDS-PAGE and transferred to nitrocellulose membranes. Data were normalized by the intensity of a-tubulin–derived signals and relatedto the value of nonglycated BSA plus control-aptamer–treated cells. n = 3/group (A–O). †P < 0.05, ††P < 0.01 compared with BSA + Con-apt;‡P < 0.05, ‡‡P < 0.01 compared with AGE + Con-apt.

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Author Contributions. T.M., Y.H., Y.N., N.N., and K.F. acquired, analyzed,and interpreted data. S.-i.Y. conceptualized and designed the study; acquired,analyzed, and interpreted data; and drafted the manuscript. S.-i.Y. is the guarantorof this work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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