Original Article - Diabetes · that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy. Diabetes 55: 2510–2522, 2006 I n developed countries, diabetic

Original Article

RAGE Control of Diabetic Nephropathy in a MouseModelEffects of RAGE Gene Disruption and Administration ofLow–Molecular Weight HeparinKhin-Mar Myint,

1Yasuhiko Yamamoto,

1Toshio Doi,

2Ichiro Kato,

3Ai Harashima,

1Hideto Yonekura,

1,4

Takuo Watanabe,1

Harumichi Shinohara,5

Masayoshi Takeuchi,6

Koichi Tsuneyama,7

Noriyoshi Hashimoto,8

Masahide Asano,8

Shin Takasawa,9

Hiroshi Okamoto,9

and Hiroshi Yamamoto1

Diabetic nephropathy is a major microvascular complica-tion in long-standing diabetic patients who eventually un-dergo renal dialysis or transplantation. To preventdevelopment of this disease and to improve advancedkidney injury, effective therapies directed toward the keymolecular target are required. In this study, we examinedwhether inhibition of the receptor for advanced glycationend products (RAGE) could attenuate changes in the dia-betic kidney. Here, we show that inactivation of the RAGEgene in a mouse model of diabetic nephropathy results insignificant suppression of kidney changes, including kidneyenlargement, increased glomerular cell number, mesangialexpansion, advanced glomerulosclerosis, increased albu-minuria, and increased serum creatinine compared withwild-type diabetic mice. The degree of kidney injury wasproportional to RAGE gene dosage. Furthermore, we showthat low–molecular weight heparin (LMWH) can bind

RAGE at a mean equilibrium dissociation constant (Kd)value of �17 nmol/l and act as an antagonist to RAGE.LMWH treatment of mice significantly prevented albumin-uria and increased glomerular cell number, mesangial ex-pansion, and glomerulosclerosis in a dose-dependentmanner; it also significantly improved the indexes of ad-vanced-stage diabetic nephropathy. This study providesinsight into the pathological role of RAGE in both early-and advanced-phase diabetic nephropathy and suggeststhat RAGE antagonists will be a useful remedy in thetreatment of diabetic nephropathy. Diabetes 55:2510–2522, 2006

In developed countries, diabetic nephropathy is themost common cause of end-stage renal disease(ESRD) (1), affecting �40% of diabetic patients (2).The hallmark characteristics of this disease are

persistent albuminuria and progressive expansion of themesangial matrix in its early phase. These lesions lead tothe development of glomerulosclerosis, which in turndestroys the renal filtration unit and eventually causesrenal failure (3). To understand the pathogenesis of dia-betic nephropathy and to develop preventive and thera-peutic means, causal molecular accounts for this diseasehave been investigated.

In diabetes, prolonged hyperglycemia drives glycationreaction and nonenzymatic cross-linking between proteinsand glucose or its derivatives. A series of further complexmolecular rearrangements yield irreversible advanced gly-cation end products (AGEs) (4). AGEs have been knownas the major factors that contribute to the pathogenesis ofdiabetes complications (5). In AGE-induced tissue damageand dysfunction, the receptor for AGEs (RAGE)-depen-dent mechanisms are likely to be responsible (6,7). Our invivo study in RAGE-overexpressing diabetic mice revealedthe functional importance of the AGE-RAGE system in thedevelopment of diabetic nephropathy (8). These micemodels developed advanced glomerulosclerosis, occasion-ally with nodular lesions and renal insufficiency, whichresembled human diabetic nephropathy but within ashorter time period (8). We hypothesized that RAGE was akey molecular target for therapeutic intervention in dia-betic nephropathy.

From the 1Department of Biochemistry and Molecular Vascular Biology,Kanazawa University Graduate School of Medical Science, Kanazawa, Japan;the 2Department of Clinical Biology and Medicine, School of Medicine, TheUniversity of Tokushima, Tokushima, Japan; the 3Department of Biochemis-try, Faculty of Medicine, University of Toyama, Toyama, Japan; the 4Depart-ment of Biochemistry, Kanazawa Medical University, Ishikawa, Japan; the5Department of Anatomy, Kanazawa Medical University, Ishikawa, Japan; the6Department of Pathophysiological Science, Faculty of Pharmaceutical Sci-ence, Hokuriku University, Kanazawa, Japan; the 7Department of Pathology,Faculty of Medicine, University of Toyama, Toyama, Japan; the 8Division ofTransgenic Animal Science, Kanazawa University Advanced Science ResearchCenter, Kanazawa, Japan; and the 9Department of Advanced BiologicalSciences for Regeneration (Kotobiken Medical Laboratories), Tohoku Univer-sity Graduate School of Medicine, Sendai, Japan.

Address correspondence and reprint requests to Hiroshi Yamamoto, MD,PhD, Department of Biochemistry and Molecular Vascular Biology, KanazawaUniversity Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa920-8640, Japan. E-mail: [email protected].

Received for publication 16 February 2006 and accepted in revised form 15June 2006.

K.-M.M. and Y.Y. contributed equally to this work.AGE, advanced glycation end product; CML, Nε-carboxymethyl-lysine;

CTGF, connective tissue growth factor; ELISA, enzyme-linked immunosor-bent assay; ESRD, end-stage renal disease; GAG, glycosaminoglycan; GAPDH,glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical veinendothelial cell; iNOS, inducible nitric oxide synthase; LMWH, low–molecularweight heparin; NF�B, nuclear factor-�B; PAM, periodic acid-methenaminesilver; PAS, periodic acid Schiff; RAGE, receptor for AGEs; siRNA, smallinterfering RNA; SPR, surface plasmon resonance; VCAM, vascular celladhesion molecule; VEGF, vascular endothelial growth factor.

DOI: 10.2337/db06-0221© 2006 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

2510 DIABETES, VOL. 55, SEPTEMBER 2006

Although Wendt et al. (9) recently reported that anenlargement of the kidney, an early-stage marker fornephropathy, was suppressed in streptozotocin-induceddiabetic RAGE-null mice, the link of RAGE to full-stagediabetic nephropathy has been unclear.

To test our hypothesis, we examined whether a deletionof RAGE would inhibit the development of kidney lesionsin another mouse model, which shows both early andadvanced stages of diabetic nephropathy, and found thatRAGE deletion ameliorated the indexes of advanced andearly stage. Furthermore, we report for the first time thatlow–molecular weight heparin (LMWH) had an antagonis-tic action on RAGE and exerted preventive and therapeu-tic effects in murine diabetic nephropathy.

RESEARCH DESIGN AND METHODS

Generation of RAGE-null mice and induction of diabetes. To performgene targeting in embryonic stem cells, we constructed a targeting vectorcontaining two loxP sites flanking the neo cassette in intron 2 and anotherloxP that was inserted into 0.6 kilobases (kb) 5� upstream of exon 1 that hadthe translation initiation site (Fig. 1A). After gene targeting in the E14-1embryonic stem cells, we identified six targeted clones by PCR and Southernblotting with probe 1, of which two were used for further experiments. Thetwo clones containing all three loxP sites in the locus gave rise to germ linechimeras by the aggregation method (10). The resultant male chimeras weremated with female Cre-transgenic mice (CD-1 background), which transientlyexpress Cre recombinase in eggs (11). Some of the newborn mice were foundto carry the deleted allele that lacks both RAGE exons 1 and 2 and neocassette. Mutant mice were backcrossed to CD-1 for more than four genera-tions. For PCR genotyping, three primers were used: 5�-CCAGAGTGACAACAGAGCAGAC-3� (primer 1), 5�-GGTCAGAACATCACAGCCCGGA-3� (primer 2),and 5�-CCTCGCCTGTTAGTTGCCCGAC-3� (primer 3) (nucleotides 73915-73936, 74523-74544, and 74881-74902 in GenBank accession no. AF030001,respectively). To induce diabetes, the mutant RAGE (�/�) mice werecrossbred with other transgenic mice (CD-1) carrying human cDNA forinducible nitric oxide synthase (iNOS) under the control of an insulinpromoter (iNOSTg) (8,12). The resultant male iNOSTg (�) RAGE (�/�) micewere crossbred with female RAGE (�/�) mice. The resultant six groups ofmale littermates were used for analysis after PCR verification. All animalswere fed a standard mouse diet (346 kcal/100 g [protein 30.4, fat 8.6, andcarbohydrate 44.0%], Labo H Standard; NIHON NOSAN, Kanagawa, Japan).The levels of blood glucose and HbA1c (A1C) were measured from tail veinblood using a Dexter Z sensor and DCA2000 analyzer (Bayer-Medical, Tokyo,Japan) (8), respectively. The procedures were approved by the institutionalanimal care and use committee guideline at Kanazawa University.RT-PCR and Western blot analyses. Total RNA was isolated from varioustissues using a high pure RNA isolation kit (Roche Diagnostics, Basel,Switzerland) and reverse transcribed as described (8,13). The primer se-quences for mouse RAGE mRNA detection were 5�-CCTGGGTGCTGGTTCTTGCTCT-3� and 5�-GATCTGGGTGCTCTTACGGTCC-3� (nucleotides 31-52 and12091-230 in GenBank accession no. L33412); those for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA detection, human vascularendothelial growth factor (VEGF) mRNA detection, and human �-actin mRNAdetection were the same as described (8,14,15). The amounts of total RNAtemplates (100 ng) and the numbers for amplification cycles (30 cycles formouse RAGE, GAPDH, and human VEGF; 28 cycles for human �-actin) werechosen in semiquantitative ranges. An aliquot of each RT-PCR product waselectrophoresed on 2% agarose gel containing ethidium bromide. Western blotanalysis was carried out as previously described (8,16). Briefly, proteins in thetissue lysates were boiled and resolved by SDS-PAGE (12.5%) and thentransferred onto a polyvinylidene fluoride membrane (Millipore). The mem-branes were incubated with a polyclonal anti-RAGE antibody, and immuno-reacted bands were visualized with an enhanced chemiluminescencedetection system (Amarsham Pharmacia Biotech).Determination of AGE concentration. Serum Nε-carboxymethyl-lysine(CML) and non-CML AGEs were differentially determined using a competitiveenzyme-linked immunosorbent assay (ELISA) with an anti-CML antibody andan anti–non-CML AGE antibody as previously described (8,17). The non-CMLAGE antibody used in this study did not cross-react with several structurallyidentified AGE-modified BSA, including pyrraline BSA, pentosidine BSA,argpyrimidine BSA, 3-deoxyglucosone imidazolone BSA, carboxymethyllysineBSA, carboxyethyllysine BSA, glyoxal-lysine dimmer, or methyglyoxal-lysinedimmer (18); undefined AGE structures may be recognized by the non-CML

AGE antibody. CML or non-CML AGE (1 unit/ml) corresponded to a proteinconcentration of 1 �g/ml CML-BSA or non-CML AGE-BSA, respectively.Determination of urine albumin creatinine ratio and serum creatinine.

Urinary or serum creatinine was measured by the Jaffe reaction (8,19). Thelevel of urinary albumin was measured by ELISA with sheep anti-mousealbumin as described (8,20). The ratio of urinary albumin to creatinine wasthen calculated.Renal histology and morphometric analyses. Kidneys were processed forlight microscopic examination, and the severity of renal sclerosis was scoredby plural analysis on an arbitrary 0- to 4-point scale (8,21,22). The meanglomerular tuft area was determined as described previously (8). To quantifyglomerulosclerosis, periodic acid-methenamine silver (PAM) stain-positivearea in the mesangium was determined using an image analyzer with amicroscope (IPAP; Sumitomo Chemical, Osaka, Japan) (23). Immunofluores-cence analysis was carried out as previously described using an anti-CMLantibody and an anti–non-CML AGE antibody (8,24).LMWH and binding assay. We used dalteparin sodium (Fragmin; Pfizer) asa LMWH, which was produced from porcine intestine–derived heparin by thedeaminative hydrolysis with nitrous acid. Its average molecular weight is5,000. Direct binding of LMWH to RAGE was assayed by surface plasmonresonance (SPR) assay (Biacore 2000; Biacore, Uppsara, Sweden). Purifiedsecretory isoform of RAGE proteins (16), which have a ligand-binding site,were immobilized on a CM5 research-grade sensor chip according to standardamine coupling procedures (15,25) with an amine coupling kit (Biacore) to adensity of �5,500 response units (RU); this density corresponded to 110fmol/mm2. On the control surface, rabbit IgG was immobilized at the samedensity. The sample and flow buffer used was 10 mmol/l HEPES (pH 7.4), 0.15mol/l NaCl, 3 mmol/l EDTA, and 0.005% surfactant P-20, and binding wasmeasured at 25°C at a flow rate of 20 �l/min. The sensor chips wereregenerated by washing with 10 mmol/l NaOH and 0.5% SDS. The meanequilibrium dissociation constant (Kd) was measured using global fitting ofmonoexponential rate equations derived from the simple 1:1 Langmuir bindingmodel. Competitive inhibition assay by LMWH in AGE-RAGE interaction wasperformed using an AGE-BSA–coated 96-well plate method as previouslydescribed (26).Nuclear factor-�B luciferase assay. C6 rat glioma cells were used for thisassay according to a previously described procedure (25,27). Briefly, cellswere transfected with a plasmid encoding luciferase cDNA under an enhancerelement containing five nuclear factor-�B (NF�B)-binding sites (pNF�B-Luc;Stratagene) and a plasmid containing human full-length RAGE cDNA. Singlestably transfected clones were selected, and the expression of RAGE wasverified by Western blotting. A typical clone was used for subsequent analyses.After a 24-h preincubation in Dulbecco’s modified Eagle’s medium, supple-mented with 0.1% fetal bovine serum, the cells were stimulated by glyceral-dehyde-derived AGE-BSA (16) for 4 h. Endotoxin was tested with LimulusHS-test Wako (Wako Pure Chemical Industries, Osaka, Japan); endotoxin wasnot detected in the AGE preparations. The luciferase activity was determinedusing Luciferase Assay System (Promega) and measured in a luminometer(Fluoroscan Ascent FL; Labsystems). RAGE expression was silenced by thesmall interfering RNA (siRNA)-expression system using the pSilencer 3.0-H1vector (Ambion, Austin, TX). A dominat-negative RAGE lacking cytoplasmicdomain was overexpressed according to a previous report by Huttunen et al.(27).Vascular cell adhesion molecule-1 and VEGF assays. Vascular cell adhe-sion molecule (VCAM)-1 assay was performed by ELISA as described (28).Briefly, human umbilical vein endothelial cells (HUVECs) were seeded on a96-well plate and were stimulated with glyceraldehydes-derived AGE-BSA ornonglycated BSA 10 �g/ml for 16 h after a 1-h pretreatment with LMWH 0.1 or1.0 IU/ml. After the stimulation, the cells were fixed with 1% paraformaldehydefor 15 min and then blocked overnight at 4°C with 2% BSA. After a 2-hincubation with monoclonal antibodies to VCAM-1 (R&D Systems), the cellswere incubated for 2 h with 1/1,000 diluted anti-mouse IgG conjugated toalkaline phosphatase (Zymed, San Francisco, CA). Quantification was per-formed by determination of the colorimetric conversion at optical density 405nm of substrate p-nitrophenyl phosphate (PIERCE, Rockford, IL). The degreeof specific antibody binding was calculated by subtracting the mean negativevalue without primary anti–VCAM-1 antibody. For the VEGF gene assay,RAGE overexpressing ECV304 cells (the HUVEC-derived cell line) were used.These cells were stimulated with 50 �g/ml of glyceraldehyed-derived AGE-BSA for 4 h with or without LMWH pretreatment. Total RNA was isolatedusing a total RNA isolation kit (Qiagen), and VEGF gene expression wasanalyzed by quantitative RT-PCR as previously described (15).LMWH treatment of diabetic animals. For the prevention study, maleiNOSTg mice were used and classified into four groups: one group received

K.-M. MYINT AND ASSOCIATES

DIABETES, VOL. 55, SEPTEMBER 2006 2511

LMWH via osmotic pump (0.25 IU/h delivery rate; Alzet), and the other groupsreceived subcutaneous (s.c.) injection of 40 or 80 IU of LMWH and vehicle(PBS) once a day. Wild-type CD-1 littermate mice were used as the nondia-betic control. These mice were treated from 1 to 4 months of age. For thestudy of therapeutic effects of LMWH, we used 4-month-old male iNOSTgmice, which were evaluated by renal tissue biopsy to have overt diabeticnephropathy before the LMWH treatment. Plasma LMWH concentrations andactivated partial thromboplastin time were measured as described (29,30).Immunohistochemistry. Formalin-fixed, paraffin-embedded kidney blockswere cut into 4-�m-thick sections and processed for immunohistochemistryas described (31). The primary antibodies used are a rabbit polyclonal VEGF

antibody (dilution 1:50; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbittransforming growth factor-�1 polyclonal antibody (dilution 1:50; Santa CruzBiotechnology), a goat connective tissue growth factor (CTGF) polyclonalantibody (dilution 1:50; Santa Cruz Biotechnology), and a rabbit S100 poly-clonal antibody (dilution 1:400; Dako). The peroxidase-labeled polymer forrabbit polyclonal antibody (EnVision; Dako, Carpineria, CA) or goat poly-clonal antibody (Histofine Simple Stain; NICHIREI, Tokyo, Japan) was appliedas a secondary antibody. Then, color was developed with 3-3�-diaminobenzi-dine (Sigma). The sections were counterstained with Meyer’s hematoxylin andcover slipped for microscopy observation. Brown areas were judged aspositive based on the manufacturing information from Envision System.

FIG. 1. Generation and characterization of RAGE-null mice. A: Gene targeting vector and strategy. B and C: Southern blotting of embryonic stemcell DNA and mouse tail DNA digested with KpnI and BglII and hybridized to probes 1 and 2, respectively. D: PCR genotyping with primers 1–3.Bands of 301 (primers 1 and 3) and 380 (primers 2 and 3) bp were derived from mutated and wild-type alleles, respectively. �/�, wild-type; �/�,heterozygous RAGE mutant; �/�, homozygous RAGE mutant. E: RT-PCR analysis of total RNA isolated from the brain, lung, heart, kidney, andeye of wild-type mice (�/�), heterozygous (�/�), and homozygous (�/�) RAGE mutant mice for the detection of RAGE and GAPDH mRNAs. F:Western blot analysis of the lung and kidney of wild-type mice (�/�), heterozygous (�/�), and homozygous (�/�) RAGE mutant mice. Filmexposure time: 1 and 10 min for lung and kidney, respectively.

RAGE INHIBITION IMPROVES DIABETIC NEPHROPATHY


Statistical analysis. All values were expressed as the means � SE. Compar-isons among the groups were analyzed by Student’s t test or ANOVA combinedwith a multiple comparison test (Scheffe’s type). These analyses were carriedout with the use of Stat View V software (SAS Institute). P 0.05 wasconsidered significant.

RESULTS

Generation of RAGE-null mice and induction of dia-betes. To generate RAGE-null mice, a targeting vectorwas constructed (Fig. 1A) and used for isolating embry-onic stem cell clones that acquired the targeting allele byhomologous recombination (Fig. 1B). We next createdchimeric mice composed predominantly of the targetedembryonic stem cells. The DNA fragment flanked by loxPsequences was removed by a transient expression of Crerecombinase in fertilized eggs. Newborn mice were foundto carry the mutated allele that lacked RAGE exons 1 and2 and neo cassette (Fig. 1C). Crossbreeding between theRAGE (�/�) heterozygous mice resulted in the produc-tion of RAGE (�/�) homozygous mice (Fig. 1C and D).The RAGE (�/�) mice were born in accordance withMendelian laws and grew apparently normal without grossphenotypic or microscopic abnormalities. RT-PCR andWestern blot analyses, respectively, revealed the absenceof RAGE mRNA and protein production in the RAGE(�/�) mice (Fig. 1E and F), indicating that the genedisruption resulted in a null mutation of RAGE. The samegenetic mean was used to induce diabetes, as was the casewith the previously reported RAGE transgenic mice: iNOStransgenic mice, which consistently develop hypoinsuline-mic diabetes as early as 1 week after birth due to theNO-mediated selective destruction of insulin-producingpancreatic �-cells (8,12). These mice represented a stablediabetic state with enhanced AGE formation and accumu-lation. After being backcrossed for at least four genera-tions to CD-1, which is the background ensuring a stableinduction of diabetes, RAGE (�/�) mice were crossbredwith iNOSTg mice (CD-1 background). The resultant maleheterozygous iNOSTg/RAGE (�/�) were then mated withfemale RAGE (�/�) to yield six groups of littermates withthe CD-1 background. Three groups carrying the iNOStransgene developed diabetes and were designated asDM�RAGE (�/�), DM�RAGE (�/�), and DM�RAGE(�/�). The other three devoid of the iNOS transgene neverdeveloped diabetes and were designated as DM-RAGE

(�/�), DM-RAGE (�/�), and DM-RAGE (�/�). Bloodanalysis confirmed sustained hyperglycemia and high A1Clevels in the diabetic mice, but there were no significantdifferences among the three groups (Table 1). Body weightalso did not significantly vary among the three diabeticgroups at all time points tested during the 8-month obser-vation period (Table 1). To determine the levels of circu-lating AGE, serum was assayed by competitive ELISA forthe CML derivative of AGE, mainly formed by peroxida-tion, and for non-CML AGE (8,17). Serum CML and non-CML AGE levels significantly increased in the DM�RAGE(�/�) mice in comparison with those in the DM-RAGE(�/�) mice at 8 months of age, but there were nosignificant differences among the three diabetic groups(Table 1). On the other hand, serum non-CML AGE was thehighest in DM-RAGE (�/�) mice among the nondiabeticgroups, suggesting that AGE might be accumulating as theanimals age when RAGE is totally depleted (Table 1).Deletion of RAGE reduces diabetic kidney changes.

Nephromegaly, expressed as an increase in weight ratio ofthe kidney against the body, was significantly attenuatedby RAGE depletion at both 4 (Fig. 2A and B) and 8 monthsof age (Fig. 3B). Urinary albumin-to-creatinine ratio wassignificantly decreased in the DM�RAGE (�/�) micewhen compared with the DM�RAGE (�/�) mice at 4months of age (Fig. 2C). The microscopic lesions noted inthe DM�RAGE (�/�) mice consisted of glomerular cellproliferation, mesangial expansion, and glomerulosclero-sis (Figs. 2D and 3A). Quantitatively, glomerular cellnumber was significantly smaller in the DM�RAGE (�/�)mice than the DM�RAGE (�/�) mice at 4 and 8 months ofage (Figs. 2E and 3C). Quantitative evaluation of glomer-ulosclerosis by calculating the sclerosis index and PAMstain–positive area per glomerular tuft area revealed thatprogression of the sclerosis was significantly attenuated inthe DM�RAGE (�/�) mice compared with the DM�RAGE(�/�) mice (Figs. 2G and H and 3E and F); it should benoted that the values obtained with the DM�RAGE (�/�)mice were consistently intermediate to those with theDM�RAGE (�/�) and DM�RAGE (�/�) mice. At 8months, the increase in serum creatinine level becameevident in the DM�RAGE (�/�) mice; this was alsosignificantly suppressed in the DM�RAGE (�/�) andDM�RAGE (�/�) mice (Fig. 3G). In addition, we exam-

TABLE 1Diabetes-related blood indexes

GroupAge

(months) MiceBody

weight (g)Blood glucose

(mmol/l) A1C (%)CML

(units/ml)non-CML AGE

(units/ml)

DM�RAGE (�/�) 4 11 44.7 � 1.0 26.7 � 1.9 7.2 � 0.5 4.6 � 0.4 11.2 � 1.4DM�RAGE (�/�) 4 7 42.2 � 1.9 26.9 � 2.9 7.2 � 1.0 5.5 � 0.9 11.4 � 1.5DM�RAGE (�/�) 4 6 41.5 � 1.4 27.1 � 2.0 6.2 � 0.4 7.3 � 1.4* 10.1 � 1.8DM�RAGE (�/�) 4 10 44.5 � 2.0 8.05 � 0.3 3.4 � 0.1 5.6 � 0.4 8.2 � 1.1DM�RAGE (�/�) 4 13 45.1 � 0.9 9.04 � 0.2 3.4 � 0.1 6.5 � 0.5 12.6 � 0.8DM�RAGE (�/�) 4 14 47.1 � 2.3 8.65 � 0.2 3.4 � 0.1 8.2 � 0.6 11.2 � 1.0DM�RAGE (�/�) 8 11 43.6 � 0.9 27.9 � 3.2 5.9 � 1.0 8.7 � 1.0† 19.6 � 3.8‡DM�RAGE (�/�) 8 7 42.0 � 1.7 23.2 � 5.6 5.9 � 0.6 8.5 � 0.4 15.4 � 1.1DM�RAGE (�/�) 8 6 41.5 � 1.6 22.5 � 2.6 5.2 � 0.3 6.2 � 1.2 17.4 � 5.3DM�RAGE (�/�) 8 4 44.0 � 1.6 7.77 � 0.6 2.5 5.1 � 1.3 6.8 � 0.9DM�RAGE (�/�) 8 10 46.1 � 1.4 8.15 � 0.4 2.5 7.4 � 1.3 9.6 � 1.9DM�RAGE (�/�) 8 10 46.7 � 2.2 7.72 � 0.3 2.5 6.9 � 0.5 18.4 � 3.2§

Data are means � SE. Statistical analysis was performed by ANOVA. *P 0.01 compared with the CML value of DM�RAGE (�/�) at 4months of age. †P 0.01 compared with the CML value of DM�RAGE (�/�) at 8 months of age. ‡P 0.0003 compared with the non-CMLAGE value of DM�RAGE (�/�) at 8 months of age. §P 0.003 and P 0.006 compared with the non-CML AGE values of DM�RAGE (�/�)and DM�RAGE (�/�) at 8 months of age, respectively.



FIG. 2. Renal changes in the early stage of diabetic nephropathy at 4 months of age. A: Sagittal section of the kidney. Bar, 5 mm. B: Kidneyweight–to–body weight ratio. *P < 0.007; **P < 0.004. C: Albuminuria. *P < 0.02; **P < 0.01. D: PAS staining of thin kidney sections. Bar, 50 �m.E: Glomerular cell count. *P < 0.02; **P < 0.004. F: Glomerular tuft area. *P < 0.015; DM�RAGE (�/�) vs. DM�RAGE (�/�), P � 0.082. G:Sclerosis index. *P < 0.02; **P < 0.0001. H: PAM-positive area: glomerular tuft area. *P < 0.05; **P < 0.004. I: Stain for non-CML AGE and CML.Original magnification �330. J: Stain for S100. Original magnification �400. Data are means � SE. Non-DM, nondiabetic; DM, diabetic. �/�,wild-type; �/�, heterozygous RAGE mutant; �/�, homozygous RAGE mutant.



ined AGE accumulation at 4 months by immunostaining.Non-CML or CML AGE was more densely deposited in therenal glomeruli of the DM�RAGE (�/�) mice than in

those of the RAGE-null mutants (Fig. 2I). Expression ofS100 protein, one of RAGE ligands, was also the highest inthe glomeruli of DM�RAGE (�/�) but less stained in the

FIG. 3. Renal changes in the advanced stage of diabetic nephropathy at 8 months of age. A: PAS staining of thin kidney sections. Bar, 50 �m. B:Kidney weight–to–body weight ratio. *P < 0.02. C: Glomerular cell count. *P < 0.004; **P � 0.004. D: Glomerular tuft area. *P < 0.025; **P < 0.04.E: Sclerosis index. *P < 0.02; **P < 0.0007. F: PAM-positive area: glomerular area. *P < 0.04; **P < 0.001. G: Serum creatinine level. *P < 0.02.Data are means � SE. Non-DM, nondiabetic; DM, diabetic. �/�, wild-type; �/�, heterozygous RAGE mutant; �/�, homozygous RAGE mutant.



kidney of DM�RAGE (�/�) and nondiabetic animals[DM-RAGE (�/�) and DM-RAGE (�/�)] (Fig. 2J), suggest-ing that local S100-RAGE interactions might also affect thedevelopment of diabetic nephropathy. However, serumS100 levels detected by immunoblotting were not changedamong the four groups: DM-RAGE (�/�) 100 � 15.2%;DM-RAGE (�/�) 84.8 � 23.1%; DM�RAGE (�/�) 93.1 �20.3%; and DM�RAGE (�/�) 102.2 � 28.4% (data notshown). These findings suggested that the deletion ofRAGE prevented the development of diabetic kidneychanges.Antagonistic effect of LMWH on RAGE. To overcomediabetic nephropathy, RAGE would thus be a candidatemolecule worth targeting. However, no RAGE antagonistswere available. Since RAGE is a heparin-binding protein,we speculated that heparin might influence AGE-RAGEinteractions. We initially tested the unfractionated high–molecular weight heparin. It was found to inhibit AGEassociation with RAGE. Its net action on RAGE, however,was agonistic when assessed with human endothelial cellsin culture (data not shown). We next examined whetherfragmented and fractionated heparin, LMWH, can inhibitthe AGE-RAGE interaction and antagonize RAGE signal-ing. LMWH largely lost N-sulfate residues during thefragmentation by nitrous acid and its biological actionswere speculated to be different from unfractionated high–molecular weight heparin. First, we used SPR assay tocheck LMWH binding to the purified extracellular domainof RAGE. The assay revealed a direct association with theKd of 17 nmol/l (Fig. 4A). Secondly, a dose-dependentcompetitive inhibition by LMWH was demonstrated in aplate assay of AGE binding to RAGE (Fig. 4B). Thirdly, tocheck its antagonistic activity in RAGE signaling, weperformed a cellular NF�B promoter assay using RAGE-overexpressing cells. Increased NF�B promoter-drivenluciferase activity by AGE-BSA was significantly blockedby the addition of LMWH (Fig. 4C). RAGE dependency andspecificity of the NF�B activation were clearly defined byusing soluble RAGE (sRAGE), RAGE siRNA, and a domi-nant-negative RAGE lacking the intracellular domain. Fi-nally, 0.1–1.0 IU/ml of LMWH have significantly inhibitedAGE-induced VEGF mRNA upregulation in RAGE-overex-pressing ECV304 cells and AGE-induced VCAM-1 expres-sion in HUVEC (Fig. 4D and E).Preventive and therapeutic effects of LMWH on dia-betic nephropathy. We then conducted an interventionalstudy with LMWH. Mice were divided into five groups: onenondiabetic control group and four iNOSTg diabeticgroups that received LMWH infusion via an osmotic pumpand daily subcutaneous injection of 40 or 80 IU of LMWHor vehicle alone. Each group was treated and observedfrom 1 month (subclinical state of diabetic nephropathy)to 4 months (overt and advanced nephropathy state) ofage. Pharmacokinetic study of LMWH in the dose of 40 and80 IU s.c. groups showed that the maximum plasmaconcentrations of LMWH were 0.10 and 0.15 IU/ml, respec-tively, at 1 h after injection (Fig. 5A). A weekly checkshowed that plasma LMWH concentrations were 0.10–0.15and 0.05–0.10 IU/ml in the LMWH 80 and 40 IU dailyinjection groups, respectively, and 0.05–0.10 IU/ml in theLMWH osmotic pump group during the 3-month observa-tion period (Fig. 5B). LMWH treatment did not lower theblood glucose and A1C levels of each group (Fig. 5C andD). Periodic acid Schiff (PAS) staining of the kidneysshowed reduced deposition of PAS-positive materials inthe glomeruli of the LMWH-treated groups (Fig. 5F). By

quantitative evaluations, LMWH dose dependently andsignificantly reduced urinary albumin creatinine ratio (Fig.5E), glomerular tuft area (Fig. 5G), glomerular cell number(Fig. 5H), and glomerular sclerosis index (Fig. 5I) whencompared with the control diabetic group. Finally, weperformed a therapeutic study with LMWH in diabeticmice from 4 (advanced diabetic nephropathy state con-firmed by open renal biopsy) to 5 months of age. Treat-ment with LMWH 80 IU daily s.c. injection for 1 monthrevealed significant reduction in urinary albumin creati-nine ratio, mesangial expansion, and glomerulosclerosisbut not in glomerular cell count (Fig. 6A–D). Blood glu-cose and A1C levels were again not affected. LMWH 80 IUdaily s.c. treatment for 1 week did not affect urinaryalbumin creatinine ratio in diabetic RAGE-null mutants(Fig. 6E).Expression of VEGF, TGF-�, and CTGF. We examinedthe expression of VEGF, TGF-�, and CTGF proteins in thekidney by immunostaining; these factors are well knownto link to diabetic nephropathy. The VEGF was faintlyexpressed, but detected, in the glomeruli from DM�RAGE(�/�) (Fig. 7A). The TGF-� and CTGF protein expressionlevels were the highest in DM�RAGE (�/�) comparedwith DM�RAGE (�/�) and with nondiabetic animals[DM-RAGE (�/�) and DM-RAGE (�/�)] (Fig. 7B and C).LMWH treatment suppressed the expressions of TGF-�and CTGF protein in the glomeruli (Fig. 7).

DISCUSSION

Diabetic nephropathy is a chronic and severe complicationof diabetes, causing increased mortality due to ESRD.After the onset of diabetic nephropathy, albuminuria,glomerular hypertrophy, and nephromegaly appear inearly phase, followed by mesangial expansion, glomerulo-sclerosis, and increased serum creatinine in advancedphase (32). The iNOSTg diabetic mice used in this studydeveloped kidney complications, similar to those seen inhumans, when maintained on a CD-1 background and feda high-calorie diet. They exhibited albuminuria, nephro-megaly, and increased expressions of VEGF, TGF-�, andCTGF until 4 months of age; thereafter, they displayedprogressive mesangial expansion, glomerulosclerosis, andincreased serum creatinine levels (Figs. 2, 3, and 7). Asconsistent with our previous results that showed an accel-eration of diabetic nephropathy in RAGE-overexpressingmice, the disruption of the RAGE gene was found in thisstudy to ameliorate all of the early- and advanced-phaseindexes of diabetic nephropathy (Figs. 2 and 3). Moreover,the extent of this attenuation was proportional to theRAGE gene dosage (Figs. 2 and 3). Recently, administra-tion of sRAGE or of neutralizing RAGE antibody wasreported to block the early events of diabetic kidneychanges in db/db mice, an obese type 2 diabetic mousemodel that develops only early-stage nephropathy (9,33).Moreover, it was previously reported that streptozotocin-induced diabetic RAGE-null mutant mice did not displayearly events in the kidney such as nephromegaly, in-creased expression of VEGF, and TGF-� in the renalcortex (9,34). Together with these findings, our dataindicate that RAGE contributes to the full expression ofdiabetic nephropathy and should be a target for diseaseintervention. Circulating levels of AGE were essentiallyinvariant among three diabetic groups, whereas AGEdeposition in renal glomeruli was greater in theDM�RAGE (�/�) mice than in the DM�RAGE (�/�) mice



FIG. 4. Antagonistic effect of LMWH on AGE-RAGE interaction. A: Kinetic analysis by SPR of LMWH binding to the extracellular domain of RAGE.Either 4, 8, 16, 32, or 64 nmol/l LMWH was injected (1 nmol/l � 0.78 IU/ml). After the injection (t � 60 s), the mobile phase was changed backto the buffer without LMWH. Normalized successive curves are shown from the lowest to highest concentrations. B: Competitive inhibition byLMWH of AGE binding to RAGE. C: Antagonistic effect of LMWH on AGE-RAGE signaling. Western blotting and Coomassie brilliant blue (CBB)staining of C6 glioma cells used in the luciferase assay (left). Luciferase activity driven by NF�� promoter (right). AGE, 50 �g/mlglyceraldehyde-derived AGE-BSA; BSA, 50 �g/ml nonglycated BSA; dnRAGE, vector for dominant-negative RAGE lacking cytoplasmic domain;LMWH, 1 IU/ml LMWH; sRAGE, 200 �g/ml sRAGE; siRNA, RAGE siRNA vector. *P < 0.0001. D: LMWH inhibition of AGE-induced VEGF mRNAupregulation assessed by semiquantitative RT-PCR. AGE, 50 �g/ml glyceraldehyde-derived AGE-BSA; BSA, 50 �g/ml nonglycated BSA. *P < 0.05.E: LMWH inhibition of AGE-induced VCAM-1 expression in HUVEC. AGE, 10 �g/ml glyceraldehyde-derived AGE-BSA; BSA, 10 �g/ml nonglycatedBSA. *P < 0.05. Data are means � SE.



FIG. 5. Preventive effect of LMWH on diabetic nephropathy. A: Pharmacokinetic study of LMWH. F, 80 IU s.c. injection (n � 6); E, 40 IU s.c.injection (n � 6). B: Weekly plasma LMWH concentrations. F, 80 IU s.c. daily; E, 40 IU s.c. daily; f, osmotic pump. C: Blood glucose levels at 4months of age. D: A1C levels at 4 months of age. E: Albuminuria. *P < 0.03; **P < 0.015. F: PAS staining of thin kidney sections. Bar, 50 �m. G:Glomerular tuft area. *P < 0.02; **P < 0.0001. H: Glomerular cell number. *P < 0.0001. I: Sclerosis index. *P < 0.0001. C, nondiabetic controlmice (n � 3); P, diabetic mice treated by LMWH osmotic pump (n � 6); -, diabetic mice treated with vehicle (PBS; n � 10); 40, diabetic micetreated with LMWH 40 IU daily s.c. injection (n � 9); 80, diabetic mice treated with LMWH 80 IU daily s.c. injection (n � 6). Data are means �SE.



(Fig. 2I). This finding suggests that AGE-RAGE interac-tions in situ would initiate and superdrive the developmentof renal lesions. Unexpectedly, serum AGE levels in theDM-RAGE (�/�) mice were high similar to those observedin the diabetic groups (Table 1) but lacking deposition inthe glomeruli (Fig. 2I). This result suggests that in additionto being a signaling receptor, RAGE might function as aclearance receptor for circulating AGE molecules.

RAGE would seem, therefore, to be a promising targetfor overcoming diabetic nephropathy, and if a compoundwas available to antagonize the AGE action on RAGE, aneffective remedy against this disease could be developed.In this study, an SPR assay revealed that LMWH is able tobind RAGE with an affinity approximately six times higherthan AGE (i.e., �17 vs. �100 nmol/l) (Fig. 4). The mode ofaction of LMWH was antagonistic as shown by the com-petition of AGE in RAGE binding experiments and by theinhibition of RAGE-dependent NF�B activation in glioma

cells and of AGE-induced VEGF and VCAM-1 expression inendothelial cells (Fig. 4C–E). LMWH blocked amphoterin/HMGB1-RAGE signaling by LMWH binding to both ampho-terin/HMGB1 and RAGE but did not block S100B-RAGEsignaling (data not shown). This suggests that the S100-binding site may be different from the AGE-binding regionof RAGE, or LMWH may not completely block the S100binding to RAGE. When LMWH was administered to thediabetic nephropathy mouse model, both preventive andtherapeutic effects were noted by the evaluation of therepresentative indexes of diabetic nephropathy such asalbuminuria, mesangial expansion, and glomerulosclerosis(Figs. 5 and 6). These in vivo effects were achieved with0.1–0.15 IU/ml LMWH within the therapeutic range ofplasma LMWH concentrations. That is to say that thera-peutic LMWH concentrations do not cause prolongation ofactivated clotting time or serious side effects (35,36).Consistent with our findings, it was reported that oral

FIG. 6. Therapeutic effect of LMWH on diabetic nephropathy from 16 to 20 weeks of age (A–D) or 16–17 weeks of age (E). A: Weeklydetermination of urinary albumin–to-creatinine ratio. Nontreated, PBS treated; Treated, daily 80 IU LMWH treated. *P < 0.05; **P < 0.015;***P < 0.0025. B: Glomerular tuft area. *P � 0.0007. C: Glomerular sclerosis index. *P < 0.0025. D: Glomerular cell count. C, PBS treated (n �12); LMWH, daily 80 IU LMWH treated (n � 11); Post, post 4 weeks of treatment. E: Urinary albumin-to-creatinine ratio. *P < 0.02. Post, post1-week treatment of 80 IU/day LMWH; Pre, pretreatment. �/�, diabetic wild-type mice; -/-, diabetic RAGE-null mutants. Data are means � SE.



sulodexide, a mixture of glycosaminoglycans (GAGs) com-posed of 80% LMWH and 20% dermatan sulfate improvedalbuminuria in both type 1 and type 2 diabetic patients(37). The long-term administration of exogenous GAGs is

also reported to have favorable effects on both morpho-logic and functional renal abnormalities and glomerularhemodynamics in diabetic nephropathy (38). The antico-agulant effects of LMWH were speculated to contribute to

FIG. 7. Stain for VEGF (A), TGF-� (B), and CTGF (C). Originalmagnification �400. The four groups were analyzed at 4 months of age:DM�RAGE (�/�), diabetic wild type; DM-RAGE (�/�), nondiabeticwild type; DM�RAGE (-/-), diabetic RAGE-null; and DM-RAGE (-/-),nondiabetic RAGE-null mice. DM with LMWH, diabetic wild-type micetreated with 80 IU/day LMWH for 3 months; DM with PBS, diabeticwild-type mice treated with PBS.



the improvement of diabetic nephropathy by affecting themicrocirculation. Several studies have provided evidencethat heparan sulfate and heparin may interfere with sig-naling events (39). In addition, the action of a variety ofgrowth factors (e.g., fibroblast growth factor and VEGF) isknown to be regulated by binding to heparan sulfate on thecell surface or the extracellular matrix, which promotes orrestricts interactions with their signal transducing recep-tors. In vascular endothelial cell culture, LMWH is re-ported to prevent hyperglycemia-induced endothelialdysfunctions such as expressions of intercellular adhesionmolecule-1, VCAM-1, and E-selectin and translocation ofNF�B, as well as generation of intracellular reactiveoxygen species (40). However, little is known about theevidence and mechanism by which LMWH influences thecellular events as the heparan sulfate or heparin does. Thisstudy suggested that the antagonistic effect of LMWH onRAGE at least partly contributed to the improvement ofdiabetic nephropathy. Immunohistochemically reducedS100 protein staining was observed in the LMWH-treateddiabetic mice compared with control diabetic mice (datanot shown); this may be suggesting another action ofLMWH. The protective effects of LMWH and GAGs havebeen ascribed to the inhibition of the diabetes-inducedupregulation of renal TGF-� (41). We observed that LMWHtreatment could inhibit AGE-induced TGF-� upregulationin cultured mesangial cells from wild-type mice but not inthose from RAGE-null mice (data not shown). As LMWH isalso known to be cleared principally by the renal route(42), it may help facilitate the renoprotection from dia-betic insult.

In conclusion, this study firmly established RAGE as apromising target, and RAGE antagonists will be a usefulremedy for both prophylaxis and treatment of diabeticnephropathy.

ACKNOWLEDGMENTS

This work was supported in part by the “Research for theFuture” Program of the Japan Society for the Promotion ofScience (grant 97L00805); by grants-in-aid from the Minis-try of Education, Science, Sports, Culture and Technology,Japan (no. 13670113) and for scientific research from theJapan Society for the Promotion of Sciences (nos.16790183, 17590241, and 16570113); from the Japan Diabe-tes Foundation; and from the Sankyo Foundation of LifeScience.

We thank S. Matsudaira and R. Kitamura (KanazawaUniversity) and H. Hatta (University of Toyama) for theirassistance.

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Original Article - Diabetes · that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy. Diabetes 55: 2510–2522, 2006 I n developed countries, diabetic