RAGE-Aptamer Blocks the Development and Progression of Experimental Diabetic Nephropathy

Diabetes has been an increasing global health challenge (1). Among various complications, diabetic nephropathy is the most common cause of end-stage renal disease and often is associated with an increased risk for cardiovascular disease, both of which could account for the high mortality rate in patients with diabetes (2).

Over a course of days to weeks, reducing sugars can react nonenzymatically with the amino groups of proteins and lipids to initiate a complex series of rearrangement, dehydration, and condensation and then to form a class of irreversibly cross-linked adducts called advanced glycation end products (AGEs) (3,4). The formation and accumulation of AGEs in various tissues progress under diabetic conditions (3,4). Interaction of AGEs with the receptor for AGEs (RAGE) generates oxidative stress and stimulates inflammatory and fibrotic reactions, which could contribute to progressive alteration in renal architecture and impairment of renal function in diabetes (5–8). Moreover, RAGE-overexpressing diabetic mice exhibit progressive glomerulosclerosis with renal dysfunction, whereas homozygous RAGE knockout diabetic mice show reduced mesangial matrix expansion and glomerular basement membrane thickening and sclerosis with preserved renal function (9–11). These findings suggest that suppression of the AGE-RAGE axis in the kidney may be a therapeutic target for diabetic nephropathy. Indeed, inhibitors of AGE formation, as well as blockade of the interaction of AGEs with RAGE by an exogenously administered soluble form of RAGE, attenuate the development and progression of experimental diabetic nephropathy (11–14). Long-term treatment with neutralizing RAGE antibodies reduces urinary albumin excretion (UAE) and increases creatinine clearance in both type 1 and type 2 diabetic mice (15,16). However, randomized, double-blind, placebo-controlled trials have revealed that AGE formation inhibitors, such as aminoguanidine and vitamin B6, do not significantly prevent the progression of diabetic nephropathy (17,18). Therefore, to develop a novel therapeutic strategy that specifically targets RAGE rather than AGEs is desired.

Aptamers are short, single-stranded DNA or RNA oligonucleotides that are selected against various kinds of target proteins by systematic evolution of ligands by exponential enrichment (SELEX) (19,20). Aptamers can take a three-dimensional structure in which conformation recognizes their cognate targets with high affinity and specificity (19,20). The wide range of sequences and three-dimensional folding patterns of oligonucleotides allow aptamers to function like antibodies; therefore, aptamers are termed chemical antibodies (19,20). Pegaptanib (Macugen) was the first U.S. Food and Drug Administration–approved RNA aptamer directed against the vascular endothelial growth factor 165 isoform for the treatment of wet-type age-related macular degeneration (21). ARC1779 and NU172, DNA-aptamers raised against the A1 domain of von Willebrand factor and exosite I of α-thrombin, currently are undergoing phase II clinical trials (22,23). Because aptamers are potentially used as antagonists and agonists of target proteins (20), we screened high-affinity DNA aptamers directed against RAGE (RAGE-aptamers) by using SELEX in vitro, characterized the binding affinity to and antagonistic activity for RAGE, and investigated whether continuous infusion of RAGE-aptamer could reverse as well as prevent the development of diabetic nephropathy in streptozotocin-induced diabetic rats. Moreover, we examined the effects of RAGE-aptamer on reactive oxygen species (ROS) generation and inflammatory and fibrotic reactions in AGE-exposed mesangial cells.

BSA, d-glyceraldehyde, streptozotocin, and bovine insulin were purchased from Sigma-Aldrich (St. Louis, MO).

BSA (25 mg/mL) was incubated under sterile conditions with 0.1 mol/L d-glyceraldehyde at 37°C for 7 days (24). Control nonglycated BSA was incubated in the same conditions except for the absence of d-glyceraldehyde.

Human V-domain of RAGE (vRAGE) (residues 23–121) carrying a C-terminal hexahistidine tag sequence (Novagen, Darmstadt, Germany) was amplified by PCR. vRAGE was coupled to a Ni Sepharose 6 Fast Flow bead matrix (GE Healthcare, Buckinghamshire, U.K.).

A random combinatorial single-stranded DNA library with normal phosphate ester backbone oligonucleotides (80-mer) was synthesized, and selection of RAGE-aptamer was performed by using SELEX as described previously (25,26).

vRAGE solution (1 μmol/L) was incubated with 0.5 μmol/L RAGE-aptamer or control-aptamer for 30 min. Wells precoated with 1 μg AGEs overnight were incubated with the reaction mixtures for 1 h and then with anti-RAGE polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX). After 1 h, horseradish peroxidase–conjugated anti-rabbit IgG and 3,3′,5,5′-tetramethylbenzidine were added to the wells, and absorbance at 450 nm was measured.

The binding affinities of control-aptamer, RAGE-aptamer, or AGE-BSA to vRAGE were measured by using a sensitive 27-MHz quartz crystal microbalance (QCM) binding assay (Initium Inc., Kanagawa, Japan) as described previously (26).

Male 6-week-old Wistar rats (Charles River Laboratories Japan) were used. Rats received single intraperitoneal injection of streptozotocin (60 mg/kg body weight) in 10 mmol/L citrate buffer (pH 4.5). Animals with blood glucose levels >200 mg/dL 48 h later were considered diabetic. One unit bovine insulin (two times per week) was subcutaneously injected into diabetic rats to maintain life, but it was not enough to consistently reduce blood glucose levels. Nondiabetic control rats received citrate buffer alone.

At 7 weeks after the injection of streptozotocin or vehicle, from 9 control and 12 diabetic rats, 4 each were transferred to metabolic cages for urinalysis. After 24 h, the rats were killed, and blood and kidneys were obtained for NADPH oxidase activity assay, real-time RT-PCR, and biochemical, immunohistochemical, and morphological analyses. The remaining five control and eight diabetic rats received continuous intraperitoneal infusion (2 pmol/day/g body weight) of either control-aptamer or RAGE-aptamer by an osmotic minipump (Durect Co., Cupertino, CA). After another 4 weeks, blood and kidneys were obtained for the above-mentioned analyses. At 7 and 11 weeks after the injection of streptozotocin or vehicle, blood pressures were measured by a tail-cuff sphygmomanometer by using an automated system with a photoelectric sensor.

Two days after the injection of streptozotocin or vehicle, four control and four diabetic rats were killed. The remaining 8 control and 12 diabetic rats received continuous intraperitoneal infusion (0.5 pmol/day/g body weight) of either control-aptamer or RAGE-aptamer by an osmotic minipump. After 2 weeks, rats were killed, and blood and kidneys were obtained for analyses.

Albuminuria was measured with a commercially available ELISA kit (Shibayagi, Shibukawa, Japan). Serum and urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were measured with an ELISA system (Nikken Seil, Shizuoka, Japan). Other blood chemistry was analyzed with standard enzymatic methods as described previously (27). In the preliminary study, we found that albuminuria began to increase at 2 weeks and reached a maximum at 7 weeks after the induction of diabetes. Therefore, we chose this time course and treatment duration for experiments 1 and 2. All experimental procedures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the ethical committee of Kurume University School of Medicine (Kurume, Japan).

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

The kidney sections were incubated overnight at 4°C with antibodies, and the reactions were visualized with a Histofine Simple Stain Rat MAX-PO (MULTI) kit (Nichirei, Tokyo, Japan) (27). Antibodies raised against 8-OHdG, nitrotyrosine (StressMarq Biosciences, Victoria, British Columbia, Canada), AGEs, carboxymethyllysine (CML) (TransGenic, Kobe, Japan), anti-RAGE (Santa Cruz Biotechnology), MCP-1 (Molecular Probes, Eugene, OR), intercellular adhesion molecule 1 (ICAM-1) (Abcam), vascular cell adhesion molecule 1 (VCAM-1) (Santa Cruz Biotechnology), connective tissue growth factor (CTGF) (Abcam), transforming growth factor-β (TGF-β) (Abcam), plasminogen activator 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 immunostaining and Western blot analyses. Immunohistoreactivity in five different fields in each sample was measured by cellSens version 1.14 software (Olympus, Tokyo, Japan). Three-micrometer paraffin sections were stained with Masson trichrome for light microscopic analysis as described previously (28).

RT-PCR was performed as described previously (26). Identifications 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-β, PAI-1, type I collagen, type III collagen, type IV collagen, fibronectin, 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.

Renal NADPH oxidase activity was measured by a luminescence assay as described previously (29).

Human mesangial cells were maintained in basal medium supplemented with 5% FBS (Clonetics, San Diego, CA). Mesangial cells were treated with 50 μg/mL AGE-BSA or nonglycated 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 membranes were probed with antibodies, and immune complexes were then visualized with an enhanced chemiluminescence detection system (GE Healthcare). Data were normalized by the intensity of α-tubulin–derived signals and related to the value of nonglycated BSA plus control-aptamer–treated cells. For the adhesion assay, mesangial cells were incubated with BCECF, AM–labeled THP-1 cells, and then fluorescent intensities of the adherent THP-1 cells were measured (29).

All data are presented as mean ± SD. ANOVA followed by Student t test was performed for statistical comparisons. P < 0.05 was considered significant.

In this study, 50 clones were sequenced from the pool of selected single-stranded DNAs to obtain 10 unique sequences. Structural analysis revealed that all the aptamers had a bulge-loop structure. Although all the clones significantly inhibited the binding of AGEs to vRAGE, the representative 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 to vRAGE by ∼30%. Because 100 nmol/L clone #1 did not exert a cytotoxic effect on human cultured endothelial cells (data not shown), we used it for the next experiments. We confirmed that AGE-BSA binds to vRAGE by a QCM binding assay (Fig. 1B). A sensitive 27-MHz QCM also revealed that RAGE-aptamer binds to vRAGE with a K_d of 5.68 nmol/L (Fig. 1C and Table 1). As shown in Fig. 1D, control-aptamer did not bind to vRAGE, and alteration of the sequence of aptamer additions led to similar results. Moreover, in the presence of RAGE-aptamer, AGE-BSA was not able to bind to vRAGE (Fig. 1E), which contrasts the case without RAGE-aptamer (Fig. 1B). The structure of clone #1 RAGE-aptamer used here is shown in Fig. 1F.

As shown in Fig. 1G, when RAGE-aptamer was continuously administrated for up to 7 days, it was distributed mainly in the aorta, eyes, testis, kidneys, and heart. After stopping the injection, RAGE-aptamer levels gradually decreased, but the aptamer was still detected in the aorta and kidneys at day 3 after the removal of the osmotic pump (Fig. 1H). The elimination half-life of RAGE-aptamer in the kidney was ∼4.4 ± 3.2 days.

We first examined whether RAGE-aptamer treatment could inhibit the progression of experimental diabetic nephropathy. As shown in Fig. 2A, diabetic rats at 7 weeks or age-matched control rats received continuous intraperitoneal infusion of either control-aptamer or RAGE-aptamer for another 4 weeks. Clinical characteristics of animals are shown in Table 2. Fasting blood glucose, glycated hemoglobin (HbA_1c), blood urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and ratios of kidney, liver, and heart weight to body weight in 13- and 17-week-old diabetic rats were higher than those of control rats of the same ages, whereas body weight and heart rate in these rats were lower than in control rats (Table 2). Four-week RAGE-aptamer infusion did not affect these parameters of 17-week-old diabetic rats except for the ratio of kidney weight to body weight; the increased ratio of kidney weight to body weight was significantly reduced by the treatment with RAGE-aptamer.

As shown in Fig. 2B–H, gene expression levels of components of NADPH oxidase, such as p22phox, Nox1, gp91phox, Nox4, p47phox, and p67phox, and its enzymatic activity were increased in 17-week-old diabetic rats compared with age-matched control rats, all of which except for p67phox were significantly inhibited by treatment with a continuous infusion of RAGE-aptamer. Furthermore, compared with control rats of the same age, serum 8-OHdG as well as renal levels of 8-OHdG and nitrotyrosine, oxidative stress markers, and AGE, CML, and RAGE levels in the kidneys of 17-week-old diabetic rats were significantly increased (Fig. 2I–O). RAGE-aptamer treatment for 4 weeks also inhibited increases in these parameters in diabetic rats.

As shown in Fig. 3A–T, renal mRNA and protein levels of MCP-1; ICAM-1; VCAM-1; CTGF; TGF-β; 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 significantly inhibited increases in macrophage infiltration into the 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 pivotal transcription factor that is exclusively expressed in podocytes, and podocin, a protein component of the filtration slits of podocytes (30), were significantly restored by the treatment with RAGE-aptamer (Fig. 3W and X). Whereas UAE levels were significantly higher in 13- and 17-week-old diabetic rats compared with age-matched control rats, RAGE-aptamer treatment for 4 weeks significantly reduced the UAE levels in 17-week-old diabetic rats (Fig. 3Y).

We further examined whether RAGE-aptamer treatment could block the development of experimental diabetic nephropathy. As shown in Fig. 4A, 6-week-old diabetic rats or age-matched control rats received continuous intraperitoneal infusion of either control-aptamer or RAGE-aptamer for 2 weeks. Clinical characteristics of animals are shown in Table 3. Fasting blood glucose, AST, and ALT in 8-week-old diabetic rats were higher than those of age-matched control rats, whereas body weight, heart rate, HDL cholesterol, and creatinine in these diabetic rats were lower than in the control rats (Table 3). No significant differences of anthropometric and biochemical markers were found between 8-week-old diabetic rats receiving control-aptamer or RAGE-aptamer.

As shown in Fig. 4B–I, urinary 8-OHdG excretion, nitrotyrosine immunostaining, AGE, CML, RAGE, and MCP-1 levels in the kidneys of 8-week-old diabetic rats were significantly increased compared with control rats, all of which were attenuated with the treatment with RAGE-aptamer.

As shown in Fig. 4J and K, RAGE-aptamer treatment for 2 weeks significantly restored the decrease in immunostaining levels of WT-1 and podocin in diabetic kidneys. UAE levels of 8-week-old diabetic rats were significantly increased compared with control rats, which were attenuated by the treatment with RAGE-aptamer (Fig. 4L).

Fifty micrograms per milliliter AGE-BSA for 4 h significantly increased ROS generation (Fig. 5A). MCP-1 gene expression, RAGE, ICAM-1, VCAM-1, CTGF, type III collagen, and fibronectin mRNA and protein levels were increased at 2 days after the treatment with 50 μg/mL AGE-BSA, all of which were significantly attenuated by the treatment of 100 nmol/L RAGE-aptamer (Fig. 5B–O). RAGE-aptamer at 100 nmol/L did not affect these mRNA or protein levels in nonglycated BSA-exposed mesangial cells. Because we could not detect measurable levels of MCP-1 by Western blot analysis, we studied the effects of RAGE-aptamer on THP-1 cell adhesion to mesangial cells. As shown in Fig. 5P, 50 μg/mL AGE-BSA stimulated THP-1 cell adhesion to mesangial cells, which was inhibited by 100 nmol/L RAGE-aptamer.

We show for the first time, to our knowledge, that although continuous infusion of RAGE-aptamer for 4 weeks did not improve hyperglycemia or affect lipid parameters or blood pressure levels, it attenuated the increases in macrophage infiltration into the kidneys, glomerular ECM protein accumulation, and UAE levels of 17-week-old streptozotocin-induced diabetic rats and improved podocyte damage, which was associated with a significant reduction in renal oxidative stress, AGE, RAGE, and inflammatory and fibrotic marker levels (Figs. 2 and 3). Compared with age-matched control rats, ratio of kidney weight to body weight, glomerular ECM accumulation, and UAE levels were already increased after 7 weeks of diabetes in association with significant elevations of ICAM-1, VCAM-1, CTGF, type I collagen, and fibronectin mRNA levels in the kidneys (Fig. 3), suggesting the beneficial effects of RAGE-aptamer on established renal damage in diabetic nephropathy. Furthermore, 2-week continuous administration of RAGE-aptamer just after the induction of diabetes also significantly suppressed increases in renal nitrotyrosine, AGE, and RAGE levels and urinary 8-OHdG excretion in 8-week-old diabetic rats and decreased albuminuria in association with amelioration of podocyte damage (Fig. 4). In addition, we found that RAGE-aptamer significantly inhibited AGE-induced increases in ROS generation and upregulation of RAGE mRNA levels in mesangial cells and subsequently reduced inflammatory and fibrotic reactions. Therefore, the current study suggests that RAGE-aptamer treatment could block the development and progression of diabetic nephropathy in type 1 diabetic rats by suppressing the AGE-RAGE axis in the kidneys.

Aptamers have less immunogenicity over antibodies for blocking diverse target proteins and can be easily selected with relatively low production costs. Furthermore, because of their small sizes, aptamers can more efficiently penetrate various tissues, which in concert with the above-mentioned characteristics can improve their clinical applicability (19,20). We developed RAGE-aptamers that specifically bind to RAGE and resultantly block binding of AGEs to RAGE in vitro. Because RAGE-aptamer at 100 nmol/L did not affect ROS generation or inflammatory and fibrotic reactions in nonglycated BSA–exposed mesangial cells (Fig. 5), phosphorothioate-modified RAGE-aptamer used in the current experiments exhibited a significant RAGE-antagonistic activity without agonist-like properties. Moreover, we found that RAGE-aptamer levels were increased in the kidneys of 6-week-old Wistar rats for at least 7 days after a continuous intraperitoneal infusion. The K_d of RAGE-aptamer for RAGE was 5.68 nmol/L, and its elimination half-life was 4.4 days; RAGE-aptamer was still detected in the kidneys at 3 days after stopping the injection. These observations suggest that phosphorothioate-modified RAGE-aptamer may be quite stable in vivo, and continuous infusion of RAGE-aptamer with the use of a device like an insulin pump could become a feasible therapeutic strategy for the treatment of diabetic nephropathy.

We demonstrated that both continuous infusion of RAGE-aptamer just after the induction of diabetes and after 7 weeks of diabetes significantly decreases renal AGE, CML, and RAGE levels in diabetic rats. We have previously shown that neutralizing antibody raised against RAGE or an antioxidant inhibits AGE-induced ROS generation, redox-sensitive transcriptional factor, nuclear factor-κB activation, and upregulation of RAGE mRNA levels in mesangial cells (31), indicating that AGE-RAGE–evoked ROS generation further enhances RAGE gene expression in mesangial cells, which may make a vicious cycle (31). Renal CML accumulation and its urinary excretion levels have been suppressed in RAGE-deficient streptozotocin-induced diabetic rats (32). Moreover, DNA aptamer raised against AGEs has reduced AGE levels in the kidneys of obese and type 2 diabetic mice (28). These findings suggest a positive feedback loop between RAGE-derived ROS generation and AGE accumulation in diabetic kidneys. RAGE-aptamer could decrease renal AGE, CML, and RAGE levels in type 1 diabetic rats by breaking the crosstalk between the AGE-RAGE axis and ROS.

AGE-RAGE interaction not only causes inflammatory and fibrotic reactions in mesangial cells and diabetic kidneys but also evokes podocyte damage through NADPH oxidase–mediated ROS generation (32–39). In the current study, we found that treatment with RAGE-aptamer suppressed upregulation of mRNA levels for NADPH oxidase components and its enzymatic activity in the kidneys of 17-week-old diabetic rats, which was associated with decreased serum 8-OHdG as well as renal 8-OHdG and nitrotyrosine levels. Urinary excretion levels of 8-OHdG were increased in 8-week-old diabetic rats, which were also attenuated by treatment with RAGE-aptamer. Therefore, the findings suggest that blockade of the AGE-RAGE interaction by RAGE-aptamer inhibits inflammatory and fibrotic reactions and podocyte damage in diabetic kidneys through suppression of ROS production, which could lead to the reduction of albuminuria in type 1 diabetic rats.

Epidemiological studies have supported the concept of metabolic memory in the development and progression of vascular complications in diabetes (40–43). Because AGE accumulation could reflect cumulative hyperglycemic exposure and contribute to inflammatory and fibrotic reactions and podocyte damage in diabetic kidneys through the sustained activation of RAGE (5–8,39,44–46), the AGE-RAGE axis is supposed to play a central role in the phenomenon of metabolic memory. The observation that RAGE-aptamer treatment reduced AGE accumulation and RAGE expression levels in the kidneys even when given after established renal injury may support the clinical utility of RAGE-aptamer for the treatment of diabetic nephropathy.

We have already shown that glyceraldehyde-modified AGEs contribute to diabetic complications in animal models and that their circulating levels are correlated with endothelial dysfunction and inflammatory biomarkers in high-risk patients, including those with diabetes (27,28,36,47,48). Furthermore, in glyceraldehyde-modified AGEs bound to vRAGE with a K_d of 8.60 nmol/L (Fig. 1B), the binding affinity was three orders of magnitude stronger than that of CML, carboxyethyllysine, or methylglyoxal-derived hydroimidazolone-1 (K_d of 31.4, 27.4, and 56.7 μmol/L, respectively). For this reason, we used glyceraldehyde-modified AGEs instead of CML, carboxyethyllysine, or methylglyoxal-derived hydroimidazolone-1, which have been shown to cause diabetic complications and react with RAGE (5,35).

We used a dose of 60 mg/kg streptozotocin to induce diabetes because 50–65 mg/kg streptozotocin-induced diabetic rats have exhibited hemodynamic and structural alterations in the kidneys, a considerable part of which resembles human diabetic nephropathy (49,50). Although we cannot totally exclude the possibility that this dose may be associated with nephrotoxicity, given the pathological role of AGE-RAGE axis in diabetic nephropathy (5–8), the current study suggests that RAGE-aptamer ameliorates renal damage in streptozotocin-induced diabetic rats by blocking the interaction of AGEs with RAGE. However, because streptozotocin-induced diabetic nephropathy is not completely the same as human diabetic nephropathy, further study is needed to clarify whether RAGE-aptamer may be a therapeutic strategy for the treatment of diabetic nephropathy in humans.

Because RAGE-aptamer treatment for 2 weeks did not affect the AGE-RAGE-oxidative stress system or renal injury in nondiabetic control rats at 6 weeks old (Fig. 4), we omitted a group of RAGE-aptamer–treated controls in experiment 1. Therefore, whether RAGE-aptamer treatment for 4 weeks has an effect on 13-week-old control rats remains unclear.

We found that RAGE-aptamer treatment significantly reduces 8-OHdG and nitrotyrosine levels in the kidneys of 8- and 17-week-old diabetic rats (Figs. 2 and 4). Although the effects of RAGE-aptamer on these oxidative stress markers were modest, the data of mRNA expression of Nox subunits, NADPH oxidase activity, and mesangial cell experiments support that ROS plays a major role in the aptamer actions. The effects of RAGE-aptamer on diabetic nephropathy in NADPH oxidase component knockout mice, including gp91phox- or Nox1-deficient mice, would be interesting to investigate.

Funding. This work was supported in part by Grants-in-Aid for Scientific Research C from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant number 16K07101 to T.M.) and by The Mitsubishi Foundation (grant number 20162714 to S.-i.Y.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

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 guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.