Effective treatment of diabetic kidney disease (DKD) remains a large unmet medical need. Within the disease’s complicated pathogenic mechanism, activation of the advanced glycation end products (AGEs)–receptor for AGE (RAGE) axis plays a pivotal role in the development and progression of DKD. To provide a new therapeutic strategy against DKD progression, we developed a vaccine against RAGE. Three rounds of immunization of mice with the RAGE vaccine successfully induced antigen-specific serum IgG antibody titers and elevated antibody titers were sustained for at least 38 weeks. In addition, RAGE vaccination significantly attenuated the increase in urinary albumin excretion in streptozotocin-induced diabetic mice (type 1 diabetes model) and leptin-receptor–deficient db/db mice (type 2 diabetes model). In microscopic analyses, RAGE vaccination suppressed glomerular hypertrophy and mesangial expansion in both diabetic models and significantly reduced glomerular basement membrane thickness in streptozotocin-induced diabetic mice. Results of an in vitro study indicated that the serum IgG antibody elicited by RAGE vaccination suppressed the expression of AGE-induced vascular cell adhesion molecule 1 and intracellular adhesion molecule 1 in endothelial cells. Thus, our newly developed RAGE vaccine attenuated the progression of DKD in mice and is a promising potential therapeutic strategy for patients with DKD.
According to the World Health Organization, the global prevalence of diabetes among adults has risen dramatically from 4.7% in 1980% to 8.5% in 2014, thus representing 422 million adults with diabetes worldwide (1). Over time, diabetes leads to serious microvascular complications and consequent damage to nerves, eyes, and kidneys. Diabetic kidney disease (DKD), defined as albuminuria or a decreased glomerular filtration rate, is present in >25% of people with diabetes (2,3). DKD is the leading cause of end-stage renal disease in approximately one half of incident cases (4) and increases cardiovascular events (5) and mortality among those with diabetes (6,7). Despite current approaches to the management of hyperglycemia and pharmacologic blockade of the renin–angiotensin system, the prevalence of DKD remains high (2). Therefore, to reduce burden of DKD worldwide, agents that directly target the pathogenic mechanism of DKD are needed.
Although the complicated mechanisms that drive the development and progression of DKD remain poorly understood, prolonged hyperglycemia is well known to lead to metabolic and hemodynamic derangements that involve the activation of the advanced glycation end products (AGEs)–receptor for AGEs (RAGE) axis (8), excessive production of NADPH oxidase–derived reactive oxygen species (9), and protein kinase C activation (10). Since the identification of RAGE (11), increasing evidence over the last several decades has indicated that the interaction between AGEs and RAGE plays a pivotal role in the development and progression of DKD. In fact, inhibitors of AGE formation (12–14), agents that break cross-links within AGEs (15), and RAGE inhibitors, including monoclonal antibodies (16) and aptamers (17), all attenuate albuminuria in animal models of diabetes.
Given that DKD often progresses asymptomatically over decades and therefore must be treated with multiple prescribed medications over the long term (18), medication adherence and compliance remain important considerations in the treatment of DKD. Poor adherence to and compliance with antidiabetic medication regimens increases the risk of end-stage renal disease in patients with DKD (19). Therefore, the development of therapeutic strategies to improve adherence to and compliance with DKD treatment would be advantageous. In this regard, vaccination may be a promising approach because of its prolonged therapeutic effect and low frequency of administration.
In this study, we developed a therapeutic vaccine against DKD by targeting RAGE and demonstrated that three rounds of subcutaneous immunization with the RAGE vaccine successfully induced beneficial immune responses that attenuated the development of DKD in mice.
Research Design and Methods
Male DBA/2JJcl and BKS.Cg-+Leprdb/+Leprdb/Jcl (db/db) mice were obtained from CLEA Japan, Inc. (Tokyo, Japan) and housed with ad libitum food and water under standard 12:12-h light/dark conditions. All experiments were performed in accordance with the Institutional Guidelines on Animal Experimentation at Keio University and were approved by the Keio University Institutional Animal Care and Use Committee (approval number 16071–1; Tokyo, Japan). To generate a type 1 diabetic model, 10-week-old DBA/2J mice were intraperitoneally injected with low-dose streptozotocin (STZ; 40 mg/kg) (Sigma-Aldrich Japan, Tokyo, Japan) in 0.1 mol/L sodium citrate buffer (pH 4.5) once daily for 5 consecutive days. We used db/db mice as a model for type 2 diabetes.
Preparation of RAGE Vaccine
A RAGE partial peptide (GenPept accession number NP_031451.2; amino acids 38–44) was synthesized by Eurofins Genomics (Tokyo, Japan) and coupled to the keyhole limpet hemocyanin (KLH) protein via the cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (Thermo Fisher Scientific, Waltham, MA). To form a water-in-oil emulsion of antigens for enhancing antigen-specific immune responses, we mixed 20 μg RAGE-KLH antigen (in 100 μL aqueous solution) with 100 μL Freund’s adjuvant (Sigma-Aldrich Japan). Complete Freund’s adjuvant was used for the first immunization; incomplete Freund’s adjuvant was used for subsequent immunizations.
Male DBA/2J mice (age 4 weeks) were allocated into two groups and subcutaneously immunized with either the RAGE vaccine (20 μg RAGE-KLH antigen and Freund’s adjuvant; total volume, 200 μL/mouse) or vehicle (20 μg KLH and Freund’s adjuvant; total volume, 200 μL/mouse) on three occasions at 2-week intervals (i.e., at 4, 6, and 8 weeks of age). In addition, male db/db mice (age 10 weeks) were allocated into two groups and subcutaneously immunized at 10, 12, and 14 weeks of age with either the RAGE vaccine or vehicle in the same manner as the DBA/2J mice.
Evaluation of Antibody Titers
Serum IgG antibody titers (n = 4/group) were determined using ELISAs. Each well of a 96-microwell plate (Immulon 1B; Thermo Fisher Scientific) was coated with 1.0 μg RAGE partial peptide (amino acids 38–44) conjugated with BSA in PBS and incubated at 4°C overnight. The plate was then incubated for 1 h at 25°C with blocking buffer (PBS containing the nonionic detergent Tween 20 and 1% BSA) before adding serum samples. Sera were diluted with blocking buffer (by serially diluting serum samples with an equal volume of blocking buffer for each dilution), added to each well, and incubated for 2 h at 25°C. After the plates had been washed with PBS containing the nonionic detergent Tween 20 and 1% BSA, goat anti-mouse IgG (dilution factor 1:5,000) (SouthernBiotech, Birmingham, AL) was added to each well, and the plate was incubated for 1.5 h at 25°C. Reactions were visualized using the TMB Microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD), and the optical density at a wavelength of 450 nm (OD450) was determined. The end point titer was expressed as the reciprocal log2 of the last dilution that gave an OD450 that was 0.1 unit greater than that of the negative control (20). In addition, antigen-specific serum IgM, IgA, and IgE antibody titers were evaluated in the same manner, by using goat anti-mouse IgM, goat anti-mouse IgA, and rat anti-mouse IgE (dilution factor 1:5,000) (SouthernBiotech).
Western Blotting Analyses
RAGE partial peptide (amino acids 38–44) conjugated with BSA, recombinant full-length RAGE protein (Sino Biological, Beijing, China), and KLH protein was separated by means of SDS-PAGE (4–12% gradient gel) and transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) for Western blotting. These proteins were detected in the Western blots using serum from either KLH-treated or vaccinated mice (1:200) followed by peroxidase-conjugated anti-mouse IgG secondary antibodies (1:20,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and detected with an ImageQuant LAS 4000 Mini chemiluminescence imager (GE Healthcare Bio-Sciences KK, Tokyo, Japan).
Blood glucose was measured at 2 and 12 weeks after the injection of STZ in DBA/2J mice (i.e., at 12 and 22 weeks of age) and at 10 weeks of age in db/db mice by means of the glucose oxidase method and an automated glucose meter (GlutestAce R; Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan). Urine samples (24 h) were collected from metabolic cages at 8 weeks after the injection of STZ in DBA/2J mice (n = 10/group) and at 10 weeks of age (just before the initiation of vaccination) and at 14 weeks after the final immunization in db/db mice (n = 6/group). Urinary albumin was measured by using a direct competitive ELISA according to the manufacturer’s protocol (Albuwell M; Exocell, Inc., Philadelphia, PA). Urinary creatinine, serum creatinine, serum urea nitrogen, AST, ALT, and serum glycoalbumin were measured enzymatically.
Light Microscopic Analyses
The right kidney was removed from DBA/2J mice at 12 weeks after the injection of STZ (n = 7–9/group) and from db/db mice at 14 weeks after the final immunization (n = 5/group), fixed in 4% paraformaldehyde, embedded in paraffin blocks, and sectioned. Paraffin sections were stained with periodic acid Schiff (PAS). To determine the glomerular volume in each mouse, the cross-sectional area was measured in at least 30 consecutive unselected glomeruli. The glomerular volume [V(G)] was obtained as: V(G) = β/κ × [Ā(G)]3/2, where Ā(G) is the cross-sectional glomerular area, β = 1.38 as pertains to spheres, and κ (a distribution coefficient) is 1.10 (21). In the same glomeruli, the mesangial matrix area was calculated as the area of positive PAS staining, expressed as a percentage of the total cross-sectional glomerular area.
In addition, paraffin sections were stained with Wilms tumor 1 (WT-1) antibody (Santa Cruz Biotechnology, Inc., Dallas, TX) as a podocyte marker followed by incubation with diaminobenzidine. In each mouse, the WT-1–positive podocytes in at least 30 consecutive unselected glomeruli were counted.
Electron Microscopic Analyses
At 12 weeks after the injection of STZ, the left kidney of DBA/2J mice (n = 4/group) was fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4°C overnight and postfixed with 2% osmium tetroxide in 0.1 mol/L phosphate buffer at 4°C for 2 h. The kidney sample was dehydrated in graded ethanol solutions, infiltrated with propylene oxide, embedded in 100% resin, and then incubated at 60°C for 48 h to polymerize the resin. The polymerized resin was ultra-thin sectioned at 70 nm by using an ultramicrotome with a diamond knife, and sections were mounted on copper grids. Each section was stained with 2% uranyl acetate at room temperature for 15 min and then secondary stained with lead stain solution (Sigma-Aldrich Japan) at room temperature for 3 min. The grids were observed under a transmission electron microscope (JEM-1400Plus; JEOL, Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3,296 × 2,472 pixels) were taken by using a charge-coupled device camera (EM-14830 RUBY2; JEOL, Ltd.).
The thickness of the glomerular basement membrane (GBM) was determined using the orthogonal intercept method (22). From each mouse, we captured 32–40 digital images at ×16,800 magnification (1 image per capillary loop, 8–10 capillary loops per glomerulus, and 4 glomeruli per animal). A grid mask with 16 intercepts was applied to each image using Photoshop CS4 (Adobe Systems, San Jose, CA). GBM thickness—from the basal endothelial to the basal podocyte plasma membrane—was measured at each intercept.
Antibody Function Analyses
The ability of IgG antibodies from vehicle-treated or vaccinated mice to inhibit RAGE signaling in vitro was assessed using human umbilical vein endothelial cells (HUVECs) (PromoCell, Heidelberg, Germany). HUVECs were plated in low-serum (2% v/v) media including growth factors (ScienCell Research Laboratories, Carlsbad, CA) and cultured at 37°C in a 5% CO2 atmosphere. Serum polyclonal IgG antibodies from both vehicle-treated and vaccinated mice were obtained by binding to Protein G Sepharose (GE Healthcare Bio-Sciences KK), followed by elution with glycine-HCl (pH 2.5) and neutralization with 1 mol/l Tris-HCl (pH 9.0). Purified IgGs were added to HUVECs at 10 μg/mL, after which the cells were incubated for 2 h at 37°C and then stimulated with AGE-BSA (50 μg/mL) (Abcam, Cambridge, U.K.) or BSA (50 μg/mL) (Sigma-Aldrich Japan) for 4 h at 37°C in normoglycemic conditions partially according to the previous article (23). Total RNAs were isolated from these cells by using TRIzol reagent (Thermo Fisher Scientific); 1 μg total RNA was reverse transcribed using PrimeScript RT Master Mix (Takara Bio, Otsu, Japan). The resulting synthesized cDNA underwent quantitative PCR amplification by using SYBR Green Master Mix (Thermo Fisher Scientific); the oligonucleotide primers for this amplification are listed in Supplementary Table 1. We obtained relative mRNA expression data for vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1) by using the results of real-time quantitative PCR amplification of these target transcripts normalized against those for β-actin mRNA.
Data are expressed as mean ± SEM. Statistical comparisons between two groups were made by using ANOVA followed by an unpaired two-tailed Student t test; P values <0.05 were considered statistically significant.
Data and Resource Availability
The data sets generated or analyzed during the current study are available from the corresponding author on request.
Selection of Antigen Sequence for the RAGE Vaccine and its Antibody Response
For use as an epitope, we selected the RAGE partial peptide comprising amino acids 38–44, because it overlaps the AGE-binding site and surface-exposed region (24–26) and in light of its amino acid properties, such as hydrophilicity and charge (27); these features increase the likelihood of neutralizing antibody production. To increase its antigenicity, we coupled the RAGE partial peptide to KLH protein for use as the RAGE vaccine.
To confirm the immunogenicity of our RAGE vaccine, we subcutaneously immunized 4-week-old DBA/2J mice with three doses of vaccine, each containing 20 μg antigen. This vaccination protocol rapidly induced serum IgG antibodies against RAGE (reciprocal log2 titer 15.25 ± 0.83 at 8 weeks after the last immunization; n = 4). In addition, the anti-RAGE IgG titer increased after each vaccine dose, reached its peak at 8 weeks after the last immunization (16 weeks of age), and then gradually decreased over several months (Fig. 1A). In contrast to serum IgG levels, antigen-specific serum IgM was induced at a lower level than IgG, whereas IgA and IgE antibodies were not detected in immunized mice (Fig. 1B).
In the Western blotting analysis, sera from mice immunized with RAGE vaccine bound not only to epitopes within the vaccine antigen, but also to full-length RAGE protein (Fig. 1C). In contrast, mice immunized with the KLH vehicle only did not develop antibodies that specifically bound to either full-length RAGE protein or the vaccine antigen (Fig. 1C).
In terms of safety, we measured the levels of AST, ALT, urea nitrogen, and creatinine in serum from healthy nondiabetic mice immunized with RAGE-KLH vaccine. These levels were within normal limits, thus indicating no significant impairment of liver or kidney function in these immunized mice (Fig. 2).
Evaluation of the RAGE Vaccine in an STZ-Induced Type 1 Diabetic Mouse Model
To evaluate the renoprotective effect of the RAGE vaccine in a model of type 1 diabetes, DBA/2J mice were immunized at 4, 6, and 8 weeks of age with either RAGE vaccine (20 μg antigen) or KLH vehicle only; these mice then received low-dose STZ (to induce type 1 diabetes) at 2 weeks after the last vaccination. Blood glucose, glycoalbumin, and body weight did not differ significantly between vehicle-treated mice and vaccinated mice (Table 1). In contrast, subcutaneous immunization with the RAGE vaccine significantly attenuated albuminuria (333.6 ± 104.7 μg/mg creatinine in vaccinated mice vs. 850.6 ± 208.6 μg/mg creatinine in vehicle-treated mice; P = 0.04) (Fig. 3A) in the type 1 diabetic mouse model. In contrast, the RAGE vaccine had no significant effect on either serum creatinine or urea nitrogen levels (Fig. 3B and C).
To further confirm the protective effect of RAGE vaccine on DKD, kidneys were analyzed histologically at 12 weeks after the injection of STZ. Although immunization with RAGE vaccine had no effect on overall kidney weight (Table 1), the total glomerular volume of vaccine-treated mice (3.18 ± 0.17 μm3) was significantly less than that of vehicle-treated mice (5.28 ± 0.52 μm3; P < 0.01) (Fig. 4A). In addition to the decrease in glomerular hypertrophy, glomerular mesangial matrix expansion was attenuated in vaccine-treated mice (18.8 ± 0.8%) compared with vehicle-only controls (26.3 ± 1.6%; P < 0.01) (Fig. 4B). Vaccinated mice had significantly more WT-1–positive podocytes (8.77 ± 0.36 cells/glomerulus) than vehicle-treated mice (6.72 ± 0.27 cells/glomerulus; P < 0.01) (Fig. 4C). Furthermore, ultrastructural morphometric study indicated that GBM thickness was significantly suppressed in vaccine-treated mice (181.3 ± 1.8 nm) compared with vehicle-treated animals (215.3 ± 1.9 nm; P < 0.01) (Fig. 5).
Evaluation of the RAGE Vaccine in a Mouse Model of Type 2 Diabetes
To evaluate the renal protective effect of our RAGE vaccine in a type 2 diabetic model, leptin receptor–deficient db/db mice were immunized at 10, 12, and 14 weeks of age with either RAGE vaccine (20 μg antigen) or the KLH vehicle only. Although body weight was similar between groups, kidney weight was slightly less in vaccinated mice (Table 1). At 10 weeks of age (that is, just prior to the first vaccine dose), db/db mice were already albuminuric (Fig. 6A). Whereas the urine albumin-to-creatinine ratio progressively increased in vehicle-treated db/db mice, this increase in urine albumin was suppressed in db/db mice immunized with the RAGE vaccine (737.7 ± 116.4 μg/mg creatinine in vehicle-treated mice vs. 367.0 ± 88.2 μg/mg creatinine in vaccinated mice; P = 0.03) (Fig. 6A). However, as seen in the type 1 diabetic model, neither serum creatinine nor urea nitrogen differed between vehicle-treated and vaccine-treated groups in the type 2 diabetic model (Fig. 6B and C).
Histologic analyses of db/db mice indicated that glomerular volume was significantly less in vaccine-treated animals (3.22 ± 0.23 μm3) compared with vehicle-treated animals (5.67 ± 0.18 μm3; P < 0.01) (Fig. 7A). In addition, the glomerular mesangial matrix area was significant smaller in vaccinated mice (23.6 ± 1.0%) than vehicle-treated mice (32.7 ± 2.3%; P < 0.01) (Fig. 7B).
In Vitro Examination of Antibody Effects on RAGE
To determine whether IgG antibodies elicited by immunization with RAGE vaccine directly inhibit RAGE signaling, we performed an in vitro assay. In endothelial cells, AGEs are known to induce VCAM-1 and ICAM-1 via RAGE (23,28). In this regard, AGE-BSA increased the mRNA expression of both VCAM-1 and ICAM-1 in HUVECs (Fig. 8). In addition, the elevated VCAM-1 and ICAM-1 mRNA levels were almost completely suppressed in HUVECs incubated with IgG antibodies obtained from the sera of vaccinated mice (Fig. 8). In contrast, treatment with IgGs obtained from vehicle-treated mice did not suppress VCAM-1 and ICAM-1 expression (Fig. 8).
In this study, we developed a novel therapeutic strategy against DKD. Specifically, we generated a vaccine based on a RAGE partial peptide, and we demonstrated that immunization with this RAGE vaccine attenuated albuminuria and early renal histologic changes in two mouse models of diabetes.
DKD remains an unmet medical need of high priority due to the lack of effective treatments that can prevent or reverse disease progression. Metabolic changes associated with diabetes alter renal hemodynamics and thus promote inflammation and fibrosis via incompletely understood mechanisms (29). Among the various pathogenic mechanisms of DKD, the AGE–RAGE interaction is well known to promote the pathogenesis and progression of DKD (30,31). In fact, disruption of the RAGE gene decreases albuminuria and attenuates microscopic lesions in a diabetic mouse model (30,32). The development of a therapeutic strategy against the AGEs–RAGE axis is a recent highlight in the field of DKD treatment (12–17). The significance of our present study is its demonstration of a RAGE vaccine as a potential approach that effectively targets the AGEs–RAGE axis to prevent and treat DKD.
DKD is chronic and progressive, and long-term intensive treatment is needed to prevent the advancement of disease. However, to delay DKD progression in previous animal studies, anti-RAGE–neutralizing antibody had to be administered to mice intraperitoneally three times weekly (16), and anti-RAGE DNA aptamer needed to be infused continuously in rats (17). In terms of therapeutic adherence and compliance, vaccination is a promising approach for the treatment of DKD because of its prolonged therapeutic effect and low frequency of administration (33). In our present study, high titers of antibodies against RAGE were maintained for at least 38 weeks after three rounds of immunization (Fig. 1A), leading to the possibility of a long-term therapeutic effect against DKD. In fact, we noted beneficial effects of RAGE vaccination on histologic lesions of DKD at 14 weeks after the final immunization in mouse models of both type 1 and type 2 diabetes.
We selected a RAGE partial peptide (amino acids 38–44) as a vaccine antigen. RAGE is a cell-surface receptor for AGEs and consists of three extracellular Ig-like domains (the V, C1, and C2 domains), a transmembrane helix, and an intracellular C-terminal tail (34). Findings from an AGE–RAGE binding assay using nuclear magnetic resonance spectroscopy suggest that Cys-38 and Gly-40 are involved in binding the AGE Nε-carboxy-methyl-lysine and that Cys-38, Gly-40, Ala-41, and Lys-43 are components of the AGE–BSA interaction surface (24). Site-directed mutagenesis revealed that single-substitution mutants of Lys-43 and Lys-44 markedly decreased complex formation between RAGE and AGE–BSA (25). Therefore, the RAGE partial peptide that we used as a vaccine antigen (amino acids 38–44) likely is an important site for AGE binding.
In the current study, we evaluated the efficacy of our RAGE vaccine against DKD in two mouse models of diabetes: the low-dose STZ-induced model of type 1 diabetes and the genetically obese leptin receptor–deficient db/db mice, which are a model of type 2 diabetes. These mouse models develop albuminuria and histologic changes similar to those of early DKD in humans. However, due to the relatively short experimental period, both of these animal models usually lack the marked increases in serum creatinine (Fig. 3C and 6C) and advanced histologic lesions, such as nodular glomerulosclerosis, that are characteristic of advanced DKD in human patients (35). The short life span of animal models also makes it difficult to assess the therapeutic effects. Therefore, we focused on the preventive effect in the current study. Despite the innate difficulty of accurate DKD modeling in rodents because of their relatively short life span, low-dose STZ mice and db/db mice are the most widely accepted DKD mouse models (36).
Independent of blood glucose levels, immunization with the RAGE vaccine reduced albuminuria by 60.8% in STZ-induced diabetic mice and by 50.3% in db/db mice. The results of the current study were consistent with a previous study that demonstrated that deletion of the RAGE gene in OVE26 mice (a transgenic model of severe early-onset type 1 diabetes) decreased albuminuria by 56% compared with that in RAGE-bearing, wild-type OVE26 mice (32). In addition, the effects of RAGE vaccine on the attenuation of albuminuria were equal to or greater than those of renin-angiotensin system inhibitors (37,38), sodium–glucose cotransporter 2 inhibitors (39,40), and NE2-related factor 2 agonists (41,42).
In endothelial cells, the AGE–RAGE interaction causes enhanced formation of oxygen radicals and the release of proinflammatory cytokines and adhesion molecules (43). In the current study, AGEs increased VCAM-1 and ICAM-1 expression in cultured human endothelial cells; treatment with vaccination-induced IgG antibodies apparently interrupted the AGE–RAGE axis, consequently almost completely suppressing the increased expression of VCAM-1 and ICAM-1 (Fig. 8). Mice deficient in adhesion molecules are known to be resistant to diabetic nephropathy (44). In the in vivo condition, chronic hyperglycemia promotes the AGE formation. In contrast, in the in vitro condition, AGEs induce the expression of adhesion molecules in endothelial cells at least partially independent of glucose (11,23,28). Therefore, the renoprotective effect of RAGE vaccination, independent of its modulation of blood glucose levels, may be at least partially due to its downregulation of VCAM-1 and ICAM-1 expression.
In summary, albuminuria, glomerular hypertrophy, and mesangial expansion are characteristic of early DKD. Our RAGE vaccine markedly suppressed urinary albumin excretion and attenuated these DKD-associated histologic lesions in mouse models of both type 1 (STZ-induced) and type 2 (db/db mice) diabetes. Thus, our RAGE vaccine represents a new therapeutic strategy to slow or halt DKD progression.
This article contains supplementary material online at https://doi.org/10.2337/figshare.14791647.
Acknowledgments. ELISA experiments were performed with the aid of Collaborative Research Resources, Keio University School of Medicine.
Funding. This work was supported by JSPS KAKENHI grant JP18K16006, Takeda Science Foundation, Novartis Research Grants, and the Japan Foundation for Applied Enzymes (to T.A.).
Duality of Interest. No potential conflicts of interest relevant to this article are reported.
Author Contributions. T.A. designed the study, performed experiments, analyzed data, and wrote the manuscript. T.N., K.H., A.H., N.Y., R.N., and H.I. reviewed the manuscript. All authors provided final approval for submission. T.A. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.