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Involvement of hyperglycemia in deposition of aggregated protein in glomeruli of diabetic mice
European Journal of Pharmacology 601 (2008) 129–135
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Involvement of hyperglycemia in deposition of aggregated protein in glomeruli of diabetic mice Yasushi Hirasawa a,b, Yukari Matsui b, Shoko Ohtsu b, Kazusuke Yamane b, Tohru Toyoshi b, Kohei Kyuki b, Takayuki Sakai a, Yibin Feng c, Tadashi Nagamatsu a,⁎ a b c
Department of Pharmacobiology and Therapeutics, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan Nihon Bioresearch Inc., 6-104 Majima, Fukuju-cho, Hashima, Gifu 501-6251, Japan School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong
a r t i c l e
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Article history: Received 20 July 2007 Received in revised form 1 October 2008 Accepted 3 October 2008 Available online 17 October 2008 Keywords: Diabetes Glomeruli Alpha-glucosidase inhibitor Pioglitazone Insulin Aggregated protein KK-Ay mice
a b s t r a c t The aim of this study was to clarify the influence of hyperglycemia on the deposition of aggregated protein in the glomeruli of diabetic mice. KK-Ay mice injected with aggregated bovine serum albumin accumulated more of it in the glomeruli than did ICR mice. There were no histological alterations in the glomeruli of KK-Ay mice. KK-Ay mice given voglibose in mouse-chow for 2 weeks had significantly reduced blood glucose, glycated albumin, and hemoglobinA1C levels compared with control mice. The voglibose-treated KK-Ay mice were injected with aggregated bovine serum albumin and accumulated significantly less albumin in the glomeruli than did the control mice. Pioglitazone decreased blood glucose levels compared with the control, and reduced the glomerular deposition of aggregated albumin. Glomerular aggregated bovine serum albumin levels and blood glucose levels were reduced significantly by the injection of insulin. Six times more advanced glycation endproducts were produced from aggregated bovine albumin than from non-aggregated bovine albumin on incubation with glucose and L-lysine in vitro. Glucose-loaded ICR mice generated more advanced glycation endproducts from aggregated albumin, and had more aggregated bovine albumin in the glomeruli. It was suggested that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli even early on in diabetes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Studies have demonstrated that advanced glycation endproducts are associated with complications of diabetes mellitus, including nephropathy, arteriosclerosis, and neuropathy (Takeuchi et al., 2000; Yamagishi et al., 1997). Advanced glycation endproducts are glycated proteins which form at an accelerated pace during chronic hyperglycemia. Enlargement of the mesangial area and thickening of the glomerular capillary lumen are characteristic histopathological alterations in the glomeruli of patients with diabetic nephropathy, and are replicated in the kidneys of normal rats by administering advanced glycation endproducts (Vlassara et al., 1994). Additionally, histopathological studies have demonstrated that advanced glycation endproducts accumulate in the renal cortex, mesangial area, and glomerular basement membrane of diabetic patients (Bierhaus et al., 1998; Makita et al., 1994) and rats with streptozotocin-induced diabetes (Mitsuhashi et al., 1993). The effect of long-term metoformin treatment on the formation of advanced glycation endproducts was investigated in rats with streptozotocin-induced diabetes. The treatment decreased the amount
⁎ Corresponding author. Tel.: +81 52 832 1151; fax: +81 52 834 8780. E-mail address: [email protected] (T. Nagamatsu). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.015
of advanced glycation endproducts in the lens, sciatic nerve, and renal cortex, but not in plasma (Tanaka et al.,1999). Pyridoxamine, an inhibitor of advanced glycation endproducts, has a beneficial effect on type 2 diabetic nephropathy in KK-Ay mice (Satoh et al., 2006). Glycated albumin is detected in the blood of diabetic patients with chronic renal disease, and in the glomeruli of diabetic WBN/Kob rats (Ishizaki et al., 1987; Suzuki, 1994). Glycated albumin is, like advanced glycation endproducts, critical to the complications seen in diabetic patients (Schalkwijk et al., 1999). Recently, we clarified that large quantities of aggregated bovine serum albumin accumulated in the glomeruli of KK-Ay mice and mice with streptozotocin-induced diabetes as compared with control mice a few hours after albumin was injected (Hirasawa et al., 2006; Nagamatsu et al., 2005). We postulate that the aggregated protein reacts with glucose in the blood shortly after its injection and that glycated protein is deposited in the glomeruli of spontaneously diabetic mice. Voglibose is an α-glucosidase inhibitor and prevents the beta-cell loss in diabetic rats via a reduction of hyperglycemia (Koyama et al., 2000). Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) agonist and reverses the resistance to insulin in type 2 diabetes mellitus. Pioglitazone is effective in lowering blood glucose levels and inhibiting the glycation of protein in diabetic patients (Herz et al., 2003; Ishida et al., 2004; Rahbar et al., 2000). The KK-Ay mouse is widely used as an experimental model of
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type 2 diabetes mellitus because it exhibits hyperglycemia, glucose intolerance, hyperinsulinemia, and obesity (Okazaki et al., 2002). We investigated the effect of voglibose, pioglitazone, and insulin on the deposition of aggregated bovine serum albumin in the glomeruli of KK-Ay mice, and the effect of high glucose on levels of aggregated protein in vitro and in vivo. We demonstrate that less aggregated bovine serum albumin is deposited in the glomeruli of KK-Ay mice with low blood glucose levels as a result of pharmacological manipulation, and that under high- glucose conditions, advanced glycation endproducts are generated from aggregated protein in the shortterm. 2. Materials and methods 2.1. Animals Male KK-Ay/Ta Jcl mice (KK-Ay, 7 weeks of age) purchased from Clea Japan (Tokyo, Japan) were used in the experiments. Male ICR/Crj/ CD-1 mice (ICR, 7 weeks of age) were purchased from Charles River Japan (Tokyo, Japan). The animals were housed in an animal center kept at 20 °C–26 °C with a 12 h light–dark cycle. Water and food were given ad libitum. KK-Ay mice were divided into groups of 8, each group having similar average serum glucose levels and body weights. The experiments were performed in accordance with the Guidelines for Animal Experiments of Meijo University Faculty of Pharmacy, the Japanese Pharmacological Society, and the National Institute of Health Guide for the Care and Use of Laboratory Animals.
glomeruli and tubules. The purity of glomeruli was over 95% as observed under a light microscope. To adjust the number of glomeruli in each measurement, phosphate-buffered saline was added to the suspension. The amount of aggregated bovine serum albumin in the glomeruli was determined by enzyme-linked immunosorbent assay (ELISA), using antiBSA antibody after the glomeruli were disrupted by freezing, thawing, and sonication as previously reported (Nagamatsu et al., 2005). The linear range of the ELISA is 0.5–20 ng/100 μl of bovine serum albumin. 2.5. Measurement of glucose, glycated albumin and hemoglobin A1C (HbAlc) Non-fasting blood samples were obtained from each mouse under anesthesia with sodium pentobarbital (5 mg/ml). The plasma glucose concentration was determined using Glucose C2™ (Wako Pure Chemical Industries). The glucose concentration was obtained by measuring absorbance of the red color at 505 nm. Glycated albumin was measured with an enzymatic reaction kit (GlyAlb-OY™, Oriental Yeast, Tokyo, Japan). Optical density was measured at 600 nm and 700 nm. Additionally, the albumin in samples was measured with a kit using bromocresol purple (BCP). The amount of glycated albumin was calculated as a percentage of all the albumin. HbA1c was measured by the latex-aggregation method (Rapidia™ auto HbA1C, Fujirebio, Tokyo, Japan), with measurement of optical density at 660 nm using a standard curve for HbA1C. The procedure was performed according to the manual provided by the kit's manufacturer. 2.6. Measurement of advanced glycation endproducts
2.2. Reagents The following chemical reagents were used for experiments: voglibose and pioglitazone (Takeda Chemical, Osaka, Japan), insulin (Novolin™ R 100, Novo Nordisk Pharma, Tokyo, Japan), crystallized bovine serum albumin (Seikagaku, Tokyo, Japan), D(+)-glucose (Wako Pure Industries, Osaka, Japan), Sea Block™ (Techno Chemical Co., Tokyo, Japan), anti-AGE monoclonal antibody (Trans Genic Co., Kumamoto, Japan), iron oxide (b5 μm, Aldrich Chemical Company, Milwaukee, WI, USA), monoclonal anti-bovine serum albumin antibody (clone BSA-33), biotin-conjugated anti-mouse immunoglobulin G antibody, and L-lysine hydrochloride (Sigma Chemical, St. Louis, MO, USA), streptavidinhorseradish peroxidase (Zymed Laboratories, San Francisco, CA, USA), ABTS peroxidase, and ABTS stop solution (KPL, Gaithersburg, MD, USA). 2.3. Preparation of aggregated bovine serum albumin Aggregated bovine serum albumin was prepared according to a previously reported procedure (Nagamatsu et al., 2005). Briefly, bovine serum albumin was dissolved in physiological saline at 30 mg/ml. The bovine serum albumin solution was alkalinized to pH10 with 0.2 M NaOH and incubated at 70 °C for 20 min and at 79 °C for 15 min. After being left at room temperature, the solution was neutralized with 0.2 M HCl and centrifuged at 1510 g for 30 min at 4 °C. The amount of aggregated bovine serum albumin was determined by the Bradford method, using a Dc protein Assay kit TR (Bio Rad-Japan, Tokyo). The supernatant was stored in a freezer until used. 2.4. Measurement of aggregated bovine serum albumin in glomeruli Aggregated bovine serum albumin was injected intravenously into the tail at 0.6 mg/g body weight (Nagamatsu et al., 2005). The kidneys were then perfused through the abdominal aorta with 1 mg/ml of iron oxide in phosphate-buffered saline, removed, and minced using a lazar. Small pieces of the kidneys were pressed through a 90 μm mesh screen, and a mixture of glomeruli and tubules was collected on a 38 μm mesh screen. The glomeruli containing iron oxide were isolated using a magnet (Magical TrapperTR, Toyobo, Osaka, Japan) from the suspension of
Glucose was dissolved in phosphate-buffered saline at 600 mg/dl. The glucose solution was mixed with the aggregated bovine serum albumin solution (2:1), 0.1 M of lysine added, and the mixture sterilized with a filter. The mixture was incubated at 50 °C for 96 h (Bhatwadekar and Ghole, 2005). Advanced glycation endproducts in the mixture were then determined by spectrofluorometry at 430 nm (Jasco FP-777, Tokyo, Japan) or enzyme-linked immunosolvent assay with anti-AGE monoclonal antibody (×1000), biotin-conjugated anti-mouse immunoglobulin G (×1000), streptavidin-horseradish peroxidase, and ABTS. The optical density was measured at 405 nm, using a microplate reader. 2.7. Statistical analyses The results are expressed as means ± S.E.M. The data were analyzed with SA preclinical package version 5(SAS Institute Japan, Tokyo, Japan). Variance was analyzed with the F-test prior to the determination of significant differences in comparisons of the means between two groups. Student's t-test and Aspin–Welch's t-test were used; differences of P b 0.05 were considered significant. In a multiple group comparison test of the means, we carried out Bartlett's t-test prior to the determination of significant differences. In the case of equal variance, Dunnett's parametric test was used and in the case of unequal variance Dunnett's non-parametric test was used; differences of P b 0.05 were considered significant. 3. Results 3.1. Time course of the accumulation of aggregated bovine serum albumin in glomeruli and histology (Fig. 1A,B,C) The first experiment was performed to compare the time course of the accumulation of aggregated bovine serum albumin in the glomeruli of KK-Ay mice and ICR mice. KK-Ay mice had accumulated a similar amount of albumin as ICR mice 1 h after the injection. The amount was 2.5-fold that in ICR mice 3 h after the injection (147 ± 0.5 ng/3000 glomeruli, 53± 0.2 ng/3000 glomeruli). In ICR mice, the glomerular aggregated bovine serum albumin level was negligible 12 h after
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bovine serum albumin, and the kidneys were isolated 3 h after the injection. As shown in Fig. 2D, the vehicle-treated KK-Ay mice had 204 ng/3000 glomeruli of aggregated bovine serum albumin at 3 h. KK-Ay mice given the 0.001% voglibose-chow had 144 ng/3000 glomeruli 3 h later; the value was significantly less than that of the vehicle-treated KK-Ay mice (30% decrease). KK-Ay mice given the 0.005% voglibose-chow had 137 ng/3000 glomeruli at 3 h; the value was significantly less than that of the vehicle-treated KK-Ay mice (32% decrease).
Fig. 1. Time course of the glomerular accumulation of aggregated bovine serum albumin and glomerular histology in KK-Ay and ICR mice. (A) The kidneys were isolated under anesthesia with sodium pentobarbital at 1, 3, 6, 8, 12, 18 and 24 h after the injection of aggregated bovine serum albumin. Values are the means ± S.E.M. (n = 8). ⁎⁎P b 0.01 indicates statistically significant differences from the ICR group. Circles indicate the KK-Ay mice. Triangles indicate the ICR mice. (B) and (C) indicate representative glomeruli from KK-Ay and ICR mice, respectively. The kidneys were obtained at 9–11 weeks of age, respectively. The kidney sections were stained with hematoxylin and eosin. Original magnification is ×400.
3.1.3. Effect of long-term treatment with pioglitazone on diabetic parameters (Fig. 3A,B) We demonstrated in the first experiment that voglibose suppressed not only the increase in blood glucose levels but also the deposition of aggregated bovine serum albumin in KK-Ay mice. Because the possibility, however, remained that the effect of voglibose on the glomerular deposition of aggregated bovine serum albumin was independent of the decrease in blood glucose levels, we used pioglitazone, which has a different mechanism from that of voglibose for reducing blood glucose levels. KK-Ay mice received mouse-chow that contained 0.01% or 0.03% pioglitazone for 4 weeks. To clarify the effect of pioglitazone on diabetic syndrome, blood was obtained every week. The control KK-Ay mice had 573.3 mg/dl of glucose and 7.1% HbA1c 4 weeks after vehicle-treatment. The administration of pioglitazone to KK-Ay mice inhibited the elevation in blood glucose levels: there was a 27% decrease in the 0.01% group and a 61% decrease in the 0.03% group as compared with levels in the vehicle-treated KK-Ay group after 4 weeks. Microscopic observation showed no histological alterations in the glomeruli of KK-Ay mice of the control group or pioglitazone-treated group (data not shown). Since KK-Ay mice ate around 7 g /mouse/day of the chow in the experiment, each mouse was estimated to ingest 17.5–52.5 mg/day of pioglitazone.
injection, while in KK-Ay mice, it was 67 ± 1.1 ng/3000 glomeruli. KK-Ay mice (Fig. 1B) and ICR mice (Fig. 1C) had no histological alterations such as the thickening of capillary walls and expansion of the mesangial matrix in the glomeruli at 7 weeks of age. Levels of serum creatinine and blood urea nitrogen were in the normal range in KK-Ay mice (data not shown). 3.1.1. Effect of long-term treatment with voglibose on diabetic parameters (Fig. 2A, B, C) KK-Ay mice were given mouse-chow containing voglibose at 0.001% or 0.005% for 2 weeks. To clarify the effect of voglibose on diabetic syndrome, blood was drawn for testing. The control KK-Ay mice were found to have 359.4 mg/dl of glucose, 8.3% HbA1C, and 70.8% glycated albumin 2 weeks after the administration of vehicle. The treatment of KK-Ay mice with voglibose suppressed the rise in blood glucose levels, namely a 39% decrease in the 0.001% voglibose-treated group and a 65% decrease in the 0.005% voglibose-treated group as compared with the control KK-Ay group. HbA1C was significantly lower in voglibose-treated KK-Ay mice than vehicle-treated KK-Ay mice (40% and 54% decrease). Voglibose inhibited significantly the increase in glycated albumin in a dose-dependent manner as compared with levels in the vehicle-treated KK-Ay group (43% and 64% decrease). There were no histological alterations in the glomeruli of KK-Ay mice of the control group or voglibose-treated group. Since KK-Ay mice ate about 6 g/mouse/day of the chow in the experiment, each mouse was estimated to ingest 2–6 mg/day of voglibose. 3.1.2. Effect of long-term treatment with voglibose on deposition of aggregated bovine serum albumin in glomeruli (Fig. 2D) KK-Ay mice were given mouse-chow containing 0.001% or 0.005% voglibose for 2 weeks, followed by an i.v. injection of aggregated
Fig. 2. Effect of long-term treatment with voglibose on plasma glucose (A), hemoglobin A1c (HbA1c) (B), and glycated albumin (C) levels, and the deposition of aggregated bovine serum albumin in glomeruli (D) in KK-Ay mice. KK-Ay mice (n = 8) were given mouse-chow containing voglibose at 0.001% or 0.005% for 2 weeks. Blood samples were obtained without fasting (A, B and C). Different groups of KK-Ay mice (n = 4) were treated with voglibose (0.001% or 0.005%) for 2 weeks and were then injected with aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital 3 h after the injection of aggregated bovine serum albumin (D). The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. White columns indicate the control group. Gray columns indicate the voglibose-treated group. Values are means ± S.E.M. ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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1 unit of insulin or vehicle followed by the immediate injection of aggregated bovine serum albumin. There was no significant difference regarding glomerular levels of the aggregated protein between the insulin-treated group and the control group 1 h after injection (Fig. 4B). Insulin-treated KK-Ay mice however had significantly less aggregated bovine serum albumin in their glomeruli than did vehicle-treated KK-Ay mice (431 ng/3000 glomeruli and 615 ng/3000 glomeruli) 3 h after its injection (Fig. 4C). 3.3. Production of advanced glycation endproducts and glycated albumin in vitro (Fig. 5A,B,C)
Fig. 3. Effect of long-term treatment with pioglitazone on plasma glucose (A) and hemoglobin A1c (HbA1c) (B) levels, and the deposition of aggregated bovine serum albumin in glomeruli (C) in KK-Ay mice. KK-Ay mice (n = 8) were given mouse-chow containing pioglitazone at 0.01% or 0.03% for 4 weeks. Blood samples were obtained without fasting. Different groups of KK-Ay mice (n = 4) were treated with pioglitazone (0.01% or 0.03%) for 4 weeks and were then injected with aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital at 3 h after the injection of aggregated bovine serum albumin. The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. Circles indicate the control group. Triangles and squares indicate the 0.01% pioglitazone and 0.03% pioglitazone groups, respectively. White columns indicate the control group. Gray columns indicate the pioglitazone group. Values are means ± S.E.M. ⁎⁎P b 0.01 and ⁎ b 0.05 indicate statistically significant differences from the control group.
Bhatwadekar and Ghole (2005) demonstrated that glycation of albumin was accelerated by L-lysine and higher temperature (50 °C). We determined the amounts of advanced glycation endproducts and glycated albumin that appeared in aggregated bovine serum albumin using their procedure. The fluorescence intensity of advanced glycation endproducts was increased 2.5-fold and 3.5-fold in the 200 mg/dl and 400 mg/dl glucose groups with L-lysine, respectively, compared to those without L-lysine (Fig. 5A). When the mixture of aggregated bovine serum albumin, 600 mg/dl glucose, and L-lysine was incubated, 6 times more advanced glycation endproducts were produced than with bovine serum albumin alone, while 1.5-fold more glycated albumin appeared with bovine serum albumin than with aggregated bovine serum albumin.(Fig. 5B,C) 3.4. Effect of acute hyperglycemia on generation of advanced glycation endproducts and deposition of aggregated bovine serum albumin in glomeruli (Fig. 6A,B,C) Because insulin-treatment caused a decrease in blood glucose levels and the deposition of aggregated bovine serum albumin in KK-Ay mice,
3.1.4. Effect of long-term treatment with pioglitazone on deposition of aggregated bovine serum albumin in glomeruli (Fig. 3C) KK-Ay mice were given mouse-chow containing 0.01% or 0.03% pioglitazone for 4 weeks. The animals were then injected i.v. with aggregated bovine serum albumin, and the kidneys were isolated 3 h later. As shown in Fig. 3C, the vehicle-treated KK-Ay mice had 178 ng/ 3000 glomeruli of aggregated bovine serum albumin after 3 h. KK-Ay mice given 0.03% pioglitazone had significantly less albumin in their glomeruli than the vehicle-treated KK-Ay mice (132 ng/3000 glomeruli, 26% decrease), while 0.01% pioglitazone-treatment failed to decrease the amount of aggregated bovine serum albumin in the glomeruli. 3.2. Effect of short-term insulin-treatment on blood glucose levels and deposition of aggregated bovine serum albumin in glomeruli (Fig. 4A,B,C) We demonstrated that less aggregated protein was deposited in the glomeruli of KK-Ay mice treated with voglibose or pioglitazone than in the glomeruli of vehicle-treated KK-Ay mice. Additional experiments were done to further clarify that hyperglycemia is associated with the deposition of aggregated bovine serum albumin in diabetic glomeruli. KK-Ay mice were injected with 1 unit of insulin, and blood samples were obtained (Fig. 4A). The injection of 1 unit of insulin decreased blood glucose levels from 408 mg/dl to 182 mg/dl after 20 min while in vehicle-treated KK-Ay mice, the level was 458 mg/dl after 20 min. The blood glucose levels were still lower 200 min after the injection of insulin than the glucose levels in the vehicle-treated KK-Ay mice (Fig. 4A). KK-Ay mice were injected with
Fig. 4. Effect of short-term treatment with insulin on blood glucose levels and deposition of aggregated bovine serum albumin in glomeruli of KK-Ay mice. KK-Ay mice were injected s.c. with 1 unit of insulin, and blood was drawn to determine blood glucose levels (A). KK-Ay mice were injected s.c. with 1 unit of insulin before the injection of aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital at 1 h (B) and 3 h (C) after the injection. The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. White columns indicate the control group. Gray columns indicate the insulin group. Values are means ± S.E.M. (n = 4). ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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Fig. 5. Production of advanced glycation endproducts (AGEs) and glycated albumin (GA) with aggregated bovine serum albumin (a-BSA) in vitro. (A) The mixture of aggregated bovine serum albumin and glucose was incubated with or without L-lysine. Values are means ± S.E.M. (n = 5). ⁎⁎P b 0.01 indicates statistically significant differences from the fluorescence intensity of the 200 mg/dl glucose group and ##P b 0.01from the fluorescence intensity without L-lysine. (B) The mixture of glucose, proteins (bovine serum albumin or aggregated bovine serum albumin), and L-lysine was incubated. Levels of advanced glycation endproducts were measured by ELISA. Results are indicated as fold values of advanced glycation endproducts produced with bovine serum albumin alone. (C) Glycated albumin is indicated as a percentage of a standard glycated albumin. White columns indicate the control group, gray columns indicate the bovine serum albumin group, and striped columns the aggregated bovine serum albumin group. Values are means ± S.E.M. (n = 4). ⁎⁎P b 0.01 indicates statistically significant differences from the control group and ##P b 0.01from the bovine serum albumin.
we performed experiments to clarify whether acute hyperglycemia induces an increase in the glomerular deposition of aggregated protein and in levels of advanced glycation endproducts in the circulation. ICR mice were administered glucose solution both p.o. and i.p. several times. Blood glucose levels rose from 133 mg/dl to 521 mg/dl at 1 h, and were 640 mg/dl at 4 h after the first administration of the solution (Fig. 6A). Mice were injected with aggregated bovine serum albumin twice, as shown in Fig. 5A. Blood glucose levels were slightly decreased 1 h later (426 mg/dl) although not significantly. After the second injection of aggregated bovine serum albumin, blood glucose levels began to drop and had returned to levels similar to those in the control mice 4 h later (136 mg/dl). Levels of advanced glycation endproducts were measured in plasma obtained 4 h after the injection of aggregated bovine serum albumin. Levels of plasma advanced glycation endproducts were 1.5 times higher in the acute hyperglycemic ICR mice than in the control ICR mice (Fig. 6B). Moreover, as expected, 29% more aggregated bovine serum albumin was detected in the acute hyperglycemic ICR mice than in the control ICR mice (Fig. 6C; 36 ng/104 glomeruli and 28 ng/104 glomeruli).
in KK-Ay mice, and as a result the diabetic glomeruli could still produce additional prostaglandin E2 in response to aggregated protein. De novo prostaglandin E2 then would accelerate the
4. Discussion Previously, we demonstrated that a large amount of exogenous aggregated protein, aggregated bovine serum albumin, accumulated in the glomeruli of diabetic mice as compared with control normal mice (Hirasawa et al., 2006; Nagamatsu et al., 2005). The underlying cause of this phenomenon has yet to be determined. In the present study, we addressed the issue of whether hyperglycemia is associated with the increased deposition of aggregated protein in diabetic glomeruli through analysis of the effect on KK-Ay mice and ICR mice of pharmacological manipulations. We demonstrated that less aggregated protein was deposited in the glomeruli of KK-Ay mice in which the elevation of blood glucose levels was suppressed by long-termtreatment with voglibose or pioglitazone. While prostaglandin E2 plays a critical role in the degradation of aggregated protein in glomeruli (Nagamatsu et al., 2001), diabetic glomeruli showed no increase in the production of prostaglandin E2 in response to aggregated protein in mice with streptozotocin-induced diabetes (Hirasawa et al., 2006). Therefore, it is likely that long-term treatment with voglibose or pioglitazone retarded the development of diabetes
Fig. 6. Effect of acute hyperglycemia on the generation of advanced glycation endproducts (AGEs) in the circulation and glomerular deposition of aggregated bovine serum albumin (a-BSA). D-glucose was dissolved in distilled water at 330 mg/ml. ICR mice were administered glucose solution (3.3 mg/kg body weight) or vehicle. ICR mice were injected with aggregated bovine serum albumin (a-BSA). Blood samples were obtained via an orbital plexus vein. (A) The figure indicates time course of blood glucose. Squares indicate vehicle-treated mice (control), triangles glucose-treated mice, and circles glucose and aggregated bovine serum albumin-treated mice. (B) The figure indicates the effect of hyperglycemia on the generation of advanced glycation endproducts. (C) The figure indicates the effect of hyperglycemia on the deposition of glomeruli. The glomeruli were isolated 4 h after the first injection of aggregated bovine serum albumin. Levels of plasma advanced glycation endproducts were measured 4 h after the first injection of aggregated bovine serum albumin. White columns indicate the control group. Gray columns indicate the hyperglycemia group. Values are means ± S.E. M. (n = 8). ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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degradation of aggregated protein in diabetic glomeruli. Therefore, we performed an experiment in which a single injection of insulin was given to KK-Ay mice, to avoid the effect of long-term treatment on the kidney. The administration of insulin resulted in lower blood glucose levels and less exogenous aggregated protein in the glomeruli of KK-Ay mice. It seems unlikely that the rate of degradation of aggregated protein returned to the original level in diabetic glomeruli in such a short time after just one injection of insulin. Albumin, immunoglobulin, and advanced glycation endproducts are detected in the glomeruli in experimental models of diabetes (Axe et al., 1981; Figarola et al., 2003; Nakamura et al., 1997). Both glycated albumin and immunoglobulin are detected in the blood of diabetic patients with chronic renal disease (Chujo et al., 2006; Danze et al., 1987). It has also been demonstrated that KK-Ay mice accumulate advanced glycation endproducts, such as CML, in the glomeruli at 20 weeks of age (Ito et al., 2006; Takemoto et al., 2002). The next issue is whether the glycation of aggregated protein and advanced glycation endproducts are generated in the circulation within several hours after the injection of aggregated protein because bovine serum albumin needs to be incubated with high concentrations of glucose at 37 °C for 60 days to produce advanced glycation endproducts in vitro (Gallicchio et al., 2006). Bhatwadekar and Ghole (2005) demonstrated that nonenzymatic glycation of bovine serum albumin and the production of advanced glycation endproducts occurred in a mixture of bovine serum albumin and glucose with L-lysine after a 4 day incubation at 50 °C. In preliminary experiments, we confirmed an increase in the production of advanced glycation endproducts and glycated albumin after a 48 h incubation of aggregated bovine serum albumin as well as serum albumin according to their procedure. Furthermore, in the present study we demonstrated that aggregated serum albumin produced more advanced glycation endproducts than non-aggregated serum albumin (Fig. 5). We then attempted to clarify whether advanced glycation endproducts are generated in blood, and whether aggregated protein is deposited in greater amounts in the glomeruli of short-term hyperglycemic ICR mice. We used short-term hyperglycemia to avoid the effects of long-term hyperglycemia on kidney function. Elevated levels of advanced glycation endproducts and diminished levels of blood glucose were demonstrated in plasma after the injection of aggregated protein. Insulin plays a critical role in the lowering of blood glucose concentrations after the injection of aggregated protein because plasma insulin levels rose with the injection of aggregated protein in KK-Ay mice (Nagamatsu et al., 2005). We consider that, in addition to the effect of insulin, some blood glucose might be consumed by the generation of advanced glycation endproducts and the glycation of aggregated protein. It is worth noting that we detected more aggregated protein in the glomeruli of short-term hyperglycemic ICR mice than in the glomeruli of control mice. Therefore, it seems that the deposition of aggregated protein in glomeruli is closely connected with hyperglycemia. There are many lines of evidence that mesangial cells express scavenger receptors on their cell membrane (Brizzi et al., 2004; Skolnik et al., 1991). Scavenger receptors recognize a broad array of ligands and mediate their internalization for decomposition. Mesangial cells internalize glycated bovine albumin via advanced glycation endproduct receptor 1 (Lu et al., 2004). Yamamoto et al. (2001) demonstrated that mice overexpressing this receptor developed renal dysfunction and glomerulosclerosis when the mice were made diabetic by crossbreeding with another mouse deficient in the islet production of insulin. Taken together, it is speculated that aggregated bovine serum albumin is glycated in the circulation and forms advanced glycation endproducts, and then these products bind to scavenger receptors in glomeruli and are ingested (Tanji et al., 2000). In conclusion, we demonstrated that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli early on in diabetic mice.
References Axe, S.R., Katz, S.M., Lavine, R., 1981. Immunoglobulin deposition in the microvasculature of the streptozotocin-induced diabetic rat. Lab. Invest. 45, 229–233. Bhatwadekar, A.D., Ghole, V.S., 2005. Rapid method for the preparation of an AGE-BSA standard calibrator using thermal glycation. J. Clin. Lab. Anal. 19, 11–15. Bierhaus, A., Hofmann, M.A., Ziegler, R., Nawroth, P.P., 1998. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc. Res. 37, 586–600. Brizzi, M.F., Dentelli, P., Rosso, A., Calvi, C., Gambino, R., Cassader, M., Salvidio, G., Deferrari, G., Camussi, G., Pegoraro, L., Pagano, G., Cavallo-Perin, P., 2004. RAGE- and TGF-beta receptor-mediated signals converge on STAT5 and p21waf to control cellcycle progression of mesangial cells: a possible role in the development and progression of diabetic nephropathy. FASEB J. 18, 1249–1251. Chujo, K., Shima, K., Tada, H., Oohashi, T., Minakuchi, J., Kawashima, S., 2006. Indicators for blood glucose control in diabetics with end-stage chronic renal disease: GHb vs. glycated albumin (GA). J. Med. Investig. 53, 223–228. Danze, P.M., Tarjoman, A., Rousseaux, J., Fossati, P., Dautrevaux, M., 1987. Evidence for an increased glycation of IgG in diabetic patients. Clin. Chim. Acta 166, 143–153. Figarola, J.L., Scott, S., Loera, S., Tessler, C., Chu, P., Weiss, L., Hardy, J., Rahbar, S., 2003. LR90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia 46, 1140–1152. Gallicchio, M.A., McRobert, E.A., Tikoo, A., Cooper, M.E., Bach, L.A., 2006. Advanced glycation end products inhibit tubulogenesis and migration of kidney epithelial cells in an ezrin-dependent manner. J. Am. Soc. Nephrol. 17, 414–421. Herz, M., Johns, D., Reviriego, J., Grossman, L.D., Godin, C., Duran, S., Hawkins, F., Lochnan, H., Escobar-Jimenez, F., Hardin, P.A., Konkoy, C.S., Tan, M.H., 2003. A randomized, double-blind, placebo-controlled, clinical trial of the effects of pioglitazone on glycemic control and dyslipidemia in oral antihyperglycemic medication-naive patients with type 2 diabetes mellitus. Clin. Ther. 25, 1074–1095. Hirasawa, Y., Muramatsu, A., Suzuki, Y., Nagamatsu, T., 2006. Insufficient expression of cyclooxygenase-2 protein is associated with retarded degradation of aggregated protein in diabetic glomeruli. J. Pharmacol. Sci. 102, 173–181. Ishida, H., Takizawa, M., Ozawa, S., Nakamichi, Y., Yamaguchi, S., Katsuta, H., Tanaka, T., Maruyama, M., Katahira, H., Yoshimoto, K., Itagaki, E., Nagamatsu, S., 2004. Pioglitazone improves insulin secretory capacity and prevents the loss of betacell mass in obese diabetic db/db mice: possible protection of beta cells from oxidative stress. Metabolism 53, 488–494. Ishizaki, M., Masuda, Y., Fukuda, Y., Yamanaka, N., Masugi, Y., Shichinohe, K., Nakama, K., 1987. Renal lesions in a strain of spontaneously diabetic WBN/Kob rats. Acta Diabetol. Lat. 24, 27–35. Ito, T., Tanimoto, M., Yamada, K., Kaneko, S., Matsumoto, M., Obayashi, K., Hagiwara, S., Murakoshi, M., Aoki, T., Wakabayashi, M., Gohda, T., Funabiki, K., Maeda, K., Horikoshi, S., Tomino, Y., 2006. Glomerular changes in the KK-Ay/Ta mouse: a possible model for human type 2 diabetic nephropathy. Nephrology (Carlton) 11, 29–35. Koyama, M., Wada, R., Mizukami, H., Sakuraba, H., Odaka, H., Ikeda, H., Yagihashi, S., 2000. Inhibition of progressive reduction of islet beta-cell mass in spontaneously diabetic Goto-Kakizaki rats by alpha-glucosidase inhibitor. Metabolism 49, 347–352. Lu, C., He, J.C., Cai, W., Liu, H., Zhu, L., Vlassara, H., 2004. Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells. Proc. Natl. Acad. Sci. U. S. A. 101, 11767–11772. Makita, Z., Bucala, R., Rayfield, E.J., Friedman, E.A., Kaufman, A.M., Korbet, S.M., Barth, R.H., Winston, J.A., Fuh, H., Manogue, K.R., et al.,1994. Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure. Lancet 343, 1519–1522. Mitsuhashi, T., Nakayama, H., Itoh, T., Kuwajima, S., Aoki, S., Atsumi, T., Koike, T., 1993. Immunochemical detection of advanced glycation end products in renal cortex from STZ-induced diabetic rat. Diabetes 42, 826–832. Nagamatsu, T., Hirasawa, Y., Matsui, Y., Ohtsu, S., Yamane, K., Toyoshi, T., Kyuki, K., Suzuki, Y., 2005. Increase in the deposition of aggregated protein in the glomeruli of spontaneously diabetic mice. J. Pharmacol. Sci. 99, 287–293. Nagamatsu, T., Nagao, T., Koseki, J., Sugiura, M., Nishiyama, T., Suzuki, Y., 2001. Involvement of prostaglandin E2 in clearance of aggregated protein via protein kinase A in glomeruli. Jpn. J. Pharmacol. 85, 139–145. Nakamura, S., Makita, Z., Ishikawa, S., Yasumura, K., Fujii, W., Yanagisawa, K., Kawata, T., Koike, T., 1997. Progression of nephropathy in spontaneous diabetic rats is prevented by OPB-9195, a novel inhibitor of advanced glycation. Diabetes 46, 895–899. Okazaki, M., Saito, Y., Udaka, Y., Maruyama, M., Murakami, H., Ota, S., Kikuchi, T., Oguchi, K., 2002. Diabetic nephropathy in KK and KK-Ay mice. Exp. Anim. 51, 191–196. Rahbar, S., Natarajan, R., Yerneni, K., Scott, S., Gonzales, N., Nadler, J.L., 2000. Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation. Clin. Chim. Acta 301, 65–77. Satoh, N., Shimatsu, A., Yamada, K., Aizawa-Abe, M., Suganami, T., Kuzuya, H., Ogawa, Y., 2006. An alpha-glucosidase inhibitor, voglibose, reduces oxidative stress markers and soluble intercellular adhesion molecule 1 in obese type 2 diabetic patients. Metabolism 55, 786–793. Schalkwijk, C.G., Ligtvoet, N., Twaalfhoven, H., Jager, A., Blaauwgeers, H.G., Schlingemann, R.O., Tarnow, L., Parving, H.H., Stehouwer, C.D., van Hinsbergh, V.W., 1999. Amadori albumin in type 1 diabetic patients: correlation with markers of endothelial function, association with diabetic nephropathy, and localization in retinal capillaries. Diabetes 48, 2446–2453. Skolnik, E.Y., Yang, Z., Makita, Z., Radoff, S., Kirstein, M., Vlassara, H., 1991. Human and rat mesangial cell receptors for glucose-modified proteins: potential role in kidney tissue remodelling and diabetic nephropathy. J. Exp. Med. 174, 931–939.
Y. Hirasawa et al. / European Journal of Pharmacology 601 (2008) 129–135 Suzuki, D., 1994. Measurement of the extracellular matrix in glomeruli from patients with diabetic nephropathy using an automatic image analyzer. Nippon Jinzo Gakkai Shi 36, 1209–1215. Takemoto, M., Asker, N., Gerhardt, H., Lundkvist, A., Johansson, B.R., Saito, Y., Betsholtz, C., 2002. A new method for large scale isolation of kidney glomeruli from mice. Am. J. Pathol. 161, 799–805. Takeuchi, M., Bucala, R., Suzuki, T., Ohkubo, T., Yamazaki, M., Koike, T., Kameda, Y., Makita, Z., 2000. Neurotoxicity of advanced glycation end-products for cultured cortical neurons. J. Neuropathol. Exp. Neurol. 59, 1094–1105. Tanaka, Y., Uchino, H., Shimizu, T., Yoshii, H., Niwa, M., Ohmura, C., Mitsuhashi, N., Onuma, T., Kawamori, R., 1999. Effect of metformin on advanced glycation endproduct formation and peripheral nerve function in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 376, 17–22. Tanji, N., Markowitz, G.S., Fu, C., Kislinger, T., Taguchi, A., Pischetsrieder, M., Stern, D., Schmidt, A.M., D'Agati, V.D., 2000. Expression of advanced glycation end products
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and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease. J. Am. Soc. Nephrol. 11, 1656–1666. Vlassara, H., Striker, L.J., Teichberg, S., Fuh, H., Li, Y.M., Steffes, M., 1994. Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc. Natl. Acad. Sci. U. S. A. 91, 11704–11708. Yamagishi, S., Yonekura, H., Yamamoto, Y., Katsuno, K., Sato, F., Mita, I., Ooka, H., Satozawa, N., Kawakami, T., Nomura, M., Yamamoto, H., 1997. Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor. J. Biol. Chem. 272, 8723–8730. Yamamoto, Y., Kato, I., Doi, I., Yonekura, H., Ohashi, S., Takeuchi, M., Watanabe, T., Yamagishi, S., Sakurai, S., Takasawa, S., Okamoto, H., Yamamoto, H., 2001. Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice. J. Clin. Invest. 108, 261–268.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Involvement of hyperglycemia in deposition of aggregated protein in glomeruli of diabetic mice Yasushi Hirasawa a,b, Yukari Matsui b, Shoko Ohtsu b, Kazusuke Yamane b, Tohru Toyoshi b, Kohei Kyuki b, Takayuki Sakai a, Yibin Feng c, Tadashi Nagamatsu a,⁎ a b c
Department of Pharmacobiology and Therapeutics, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan Nihon Bioresearch Inc., 6-104 Majima, Fukuju-cho, Hashima, Gifu 501-6251, Japan School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong
a r t i c l e
i n f o
Article history: Received 20 July 2007 Received in revised form 1 October 2008 Accepted 3 October 2008 Available online 17 October 2008 Keywords: Diabetes Glomeruli Alpha-glucosidase inhibitor Pioglitazone Insulin Aggregated protein KK-Ay mice
a b s t r a c t The aim of this study was to clarify the influence of hyperglycemia on the deposition of aggregated protein in the glomeruli of diabetic mice. KK-Ay mice injected with aggregated bovine serum albumin accumulated more of it in the glomeruli than did ICR mice. There were no histological alterations in the glomeruli of KK-Ay mice. KK-Ay mice given voglibose in mouse-chow for 2 weeks had significantly reduced blood glucose, glycated albumin, and hemoglobinA1C levels compared with control mice. The voglibose-treated KK-Ay mice were injected with aggregated bovine serum albumin and accumulated significantly less albumin in the glomeruli than did the control mice. Pioglitazone decreased blood glucose levels compared with the control, and reduced the glomerular deposition of aggregated albumin. Glomerular aggregated bovine serum albumin levels and blood glucose levels were reduced significantly by the injection of insulin. Six times more advanced glycation endproducts were produced from aggregated bovine albumin than from non-aggregated bovine albumin on incubation with glucose and L-lysine in vitro. Glucose-loaded ICR mice generated more advanced glycation endproducts from aggregated albumin, and had more aggregated bovine albumin in the glomeruli. It was suggested that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli even early on in diabetes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Studies have demonstrated that advanced glycation endproducts are associated with complications of diabetes mellitus, including nephropathy, arteriosclerosis, and neuropathy (Takeuchi et al., 2000; Yamagishi et al., 1997). Advanced glycation endproducts are glycated proteins which form at an accelerated pace during chronic hyperglycemia. Enlargement of the mesangial area and thickening of the glomerular capillary lumen are characteristic histopathological alterations in the glomeruli of patients with diabetic nephropathy, and are replicated in the kidneys of normal rats by administering advanced glycation endproducts (Vlassara et al., 1994). Additionally, histopathological studies have demonstrated that advanced glycation endproducts accumulate in the renal cortex, mesangial area, and glomerular basement membrane of diabetic patients (Bierhaus et al., 1998; Makita et al., 1994) and rats with streptozotocin-induced diabetes (Mitsuhashi et al., 1993). The effect of long-term metoformin treatment on the formation of advanced glycation endproducts was investigated in rats with streptozotocin-induced diabetes. The treatment decreased the amount
⁎ Corresponding author. Tel.: +81 52 832 1151; fax: +81 52 834 8780. E-mail address: [email protected] (T. Nagamatsu). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.10.015
of advanced glycation endproducts in the lens, sciatic nerve, and renal cortex, but not in plasma (Tanaka et al.,1999). Pyridoxamine, an inhibitor of advanced glycation endproducts, has a beneficial effect on type 2 diabetic nephropathy in KK-Ay mice (Satoh et al., 2006). Glycated albumin is detected in the blood of diabetic patients with chronic renal disease, and in the glomeruli of diabetic WBN/Kob rats (Ishizaki et al., 1987; Suzuki, 1994). Glycated albumin is, like advanced glycation endproducts, critical to the complications seen in diabetic patients (Schalkwijk et al., 1999). Recently, we clarified that large quantities of aggregated bovine serum albumin accumulated in the glomeruli of KK-Ay mice and mice with streptozotocin-induced diabetes as compared with control mice a few hours after albumin was injected (Hirasawa et al., 2006; Nagamatsu et al., 2005). We postulate that the aggregated protein reacts with glucose in the blood shortly after its injection and that glycated protein is deposited in the glomeruli of spontaneously diabetic mice. Voglibose is an α-glucosidase inhibitor and prevents the beta-cell loss in diabetic rats via a reduction of hyperglycemia (Koyama et al., 2000). Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) agonist and reverses the resistance to insulin in type 2 diabetes mellitus. Pioglitazone is effective in lowering blood glucose levels and inhibiting the glycation of protein in diabetic patients (Herz et al., 2003; Ishida et al., 2004; Rahbar et al., 2000). The KK-Ay mouse is widely used as an experimental model of
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type 2 diabetes mellitus because it exhibits hyperglycemia, glucose intolerance, hyperinsulinemia, and obesity (Okazaki et al., 2002). We investigated the effect of voglibose, pioglitazone, and insulin on the deposition of aggregated bovine serum albumin in the glomeruli of KK-Ay mice, and the effect of high glucose on levels of aggregated protein in vitro and in vivo. We demonstrate that less aggregated bovine serum albumin is deposited in the glomeruli of KK-Ay mice with low blood glucose levels as a result of pharmacological manipulation, and that under high- glucose conditions, advanced glycation endproducts are generated from aggregated protein in the shortterm. 2. Materials and methods 2.1. Animals Male KK-Ay/Ta Jcl mice (KK-Ay, 7 weeks of age) purchased from Clea Japan (Tokyo, Japan) were used in the experiments. Male ICR/Crj/ CD-1 mice (ICR, 7 weeks of age) were purchased from Charles River Japan (Tokyo, Japan). The animals were housed in an animal center kept at 20 °C–26 °C with a 12 h light–dark cycle. Water and food were given ad libitum. KK-Ay mice were divided into groups of 8, each group having similar average serum glucose levels and body weights. The experiments were performed in accordance with the Guidelines for Animal Experiments of Meijo University Faculty of Pharmacy, the Japanese Pharmacological Society, and the National Institute of Health Guide for the Care and Use of Laboratory Animals.
glomeruli and tubules. The purity of glomeruli was over 95% as observed under a light microscope. To adjust the number of glomeruli in each measurement, phosphate-buffered saline was added to the suspension. The amount of aggregated bovine serum albumin in the glomeruli was determined by enzyme-linked immunosorbent assay (ELISA), using antiBSA antibody after the glomeruli were disrupted by freezing, thawing, and sonication as previously reported (Nagamatsu et al., 2005). The linear range of the ELISA is 0.5–20 ng/100 μl of bovine serum albumin. 2.5. Measurement of glucose, glycated albumin and hemoglobin A1C (HbAlc) Non-fasting blood samples were obtained from each mouse under anesthesia with sodium pentobarbital (5 mg/ml). The plasma glucose concentration was determined using Glucose C2™ (Wako Pure Chemical Industries). The glucose concentration was obtained by measuring absorbance of the red color at 505 nm. Glycated albumin was measured with an enzymatic reaction kit (GlyAlb-OY™, Oriental Yeast, Tokyo, Japan). Optical density was measured at 600 nm and 700 nm. Additionally, the albumin in samples was measured with a kit using bromocresol purple (BCP). The amount of glycated albumin was calculated as a percentage of all the albumin. HbA1c was measured by the latex-aggregation method (Rapidia™ auto HbA1C, Fujirebio, Tokyo, Japan), with measurement of optical density at 660 nm using a standard curve for HbA1C. The procedure was performed according to the manual provided by the kit's manufacturer. 2.6. Measurement of advanced glycation endproducts
2.2. Reagents The following chemical reagents were used for experiments: voglibose and pioglitazone (Takeda Chemical, Osaka, Japan), insulin (Novolin™ R 100, Novo Nordisk Pharma, Tokyo, Japan), crystallized bovine serum albumin (Seikagaku, Tokyo, Japan), D(+)-glucose (Wako Pure Industries, Osaka, Japan), Sea Block™ (Techno Chemical Co., Tokyo, Japan), anti-AGE monoclonal antibody (Trans Genic Co., Kumamoto, Japan), iron oxide (b5 μm, Aldrich Chemical Company, Milwaukee, WI, USA), monoclonal anti-bovine serum albumin antibody (clone BSA-33), biotin-conjugated anti-mouse immunoglobulin G antibody, and L-lysine hydrochloride (Sigma Chemical, St. Louis, MO, USA), streptavidinhorseradish peroxidase (Zymed Laboratories, San Francisco, CA, USA), ABTS peroxidase, and ABTS stop solution (KPL, Gaithersburg, MD, USA). 2.3. Preparation of aggregated bovine serum albumin Aggregated bovine serum albumin was prepared according to a previously reported procedure (Nagamatsu et al., 2005). Briefly, bovine serum albumin was dissolved in physiological saline at 30 mg/ml. The bovine serum albumin solution was alkalinized to pH10 with 0.2 M NaOH and incubated at 70 °C for 20 min and at 79 °C for 15 min. After being left at room temperature, the solution was neutralized with 0.2 M HCl and centrifuged at 1510 g for 30 min at 4 °C. The amount of aggregated bovine serum albumin was determined by the Bradford method, using a Dc protein Assay kit TR (Bio Rad-Japan, Tokyo). The supernatant was stored in a freezer until used. 2.4. Measurement of aggregated bovine serum albumin in glomeruli Aggregated bovine serum albumin was injected intravenously into the tail at 0.6 mg/g body weight (Nagamatsu et al., 2005). The kidneys were then perfused through the abdominal aorta with 1 mg/ml of iron oxide in phosphate-buffered saline, removed, and minced using a lazar. Small pieces of the kidneys were pressed through a 90 μm mesh screen, and a mixture of glomeruli and tubules was collected on a 38 μm mesh screen. The glomeruli containing iron oxide were isolated using a magnet (Magical TrapperTR, Toyobo, Osaka, Japan) from the suspension of
Glucose was dissolved in phosphate-buffered saline at 600 mg/dl. The glucose solution was mixed with the aggregated bovine serum albumin solution (2:1), 0.1 M of lysine added, and the mixture sterilized with a filter. The mixture was incubated at 50 °C for 96 h (Bhatwadekar and Ghole, 2005). Advanced glycation endproducts in the mixture were then determined by spectrofluorometry at 430 nm (Jasco FP-777, Tokyo, Japan) or enzyme-linked immunosolvent assay with anti-AGE monoclonal antibody (×1000), biotin-conjugated anti-mouse immunoglobulin G (×1000), streptavidin-horseradish peroxidase, and ABTS. The optical density was measured at 405 nm, using a microplate reader. 2.7. Statistical analyses The results are expressed as means ± S.E.M. The data were analyzed with SA preclinical package version 5(SAS Institute Japan, Tokyo, Japan). Variance was analyzed with the F-test prior to the determination of significant differences in comparisons of the means between two groups. Student's t-test and Aspin–Welch's t-test were used; differences of P b 0.05 were considered significant. In a multiple group comparison test of the means, we carried out Bartlett's t-test prior to the determination of significant differences. In the case of equal variance, Dunnett's parametric test was used and in the case of unequal variance Dunnett's non-parametric test was used; differences of P b 0.05 were considered significant. 3. Results 3.1. Time course of the accumulation of aggregated bovine serum albumin in glomeruli and histology (Fig. 1A,B,C) The first experiment was performed to compare the time course of the accumulation of aggregated bovine serum albumin in the glomeruli of KK-Ay mice and ICR mice. KK-Ay mice had accumulated a similar amount of albumin as ICR mice 1 h after the injection. The amount was 2.5-fold that in ICR mice 3 h after the injection (147 ± 0.5 ng/3000 glomeruli, 53± 0.2 ng/3000 glomeruli). In ICR mice, the glomerular aggregated bovine serum albumin level was negligible 12 h after
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bovine serum albumin, and the kidneys were isolated 3 h after the injection. As shown in Fig. 2D, the vehicle-treated KK-Ay mice had 204 ng/3000 glomeruli of aggregated bovine serum albumin at 3 h. KK-Ay mice given the 0.001% voglibose-chow had 144 ng/3000 glomeruli 3 h later; the value was significantly less than that of the vehicle-treated KK-Ay mice (30% decrease). KK-Ay mice given the 0.005% voglibose-chow had 137 ng/3000 glomeruli at 3 h; the value was significantly less than that of the vehicle-treated KK-Ay mice (32% decrease).
Fig. 1. Time course of the glomerular accumulation of aggregated bovine serum albumin and glomerular histology in KK-Ay and ICR mice. (A) The kidneys were isolated under anesthesia with sodium pentobarbital at 1, 3, 6, 8, 12, 18 and 24 h after the injection of aggregated bovine serum albumin. Values are the means ± S.E.M. (n = 8). ⁎⁎P b 0.01 indicates statistically significant differences from the ICR group. Circles indicate the KK-Ay mice. Triangles indicate the ICR mice. (B) and (C) indicate representative glomeruli from KK-Ay and ICR mice, respectively. The kidneys were obtained at 9–11 weeks of age, respectively. The kidney sections were stained with hematoxylin and eosin. Original magnification is ×400.
3.1.3. Effect of long-term treatment with pioglitazone on diabetic parameters (Fig. 3A,B) We demonstrated in the first experiment that voglibose suppressed not only the increase in blood glucose levels but also the deposition of aggregated bovine serum albumin in KK-Ay mice. Because the possibility, however, remained that the effect of voglibose on the glomerular deposition of aggregated bovine serum albumin was independent of the decrease in blood glucose levels, we used pioglitazone, which has a different mechanism from that of voglibose for reducing blood glucose levels. KK-Ay mice received mouse-chow that contained 0.01% or 0.03% pioglitazone for 4 weeks. To clarify the effect of pioglitazone on diabetic syndrome, blood was obtained every week. The control KK-Ay mice had 573.3 mg/dl of glucose and 7.1% HbA1c 4 weeks after vehicle-treatment. The administration of pioglitazone to KK-Ay mice inhibited the elevation in blood glucose levels: there was a 27% decrease in the 0.01% group and a 61% decrease in the 0.03% group as compared with levels in the vehicle-treated KK-Ay group after 4 weeks. Microscopic observation showed no histological alterations in the glomeruli of KK-Ay mice of the control group or pioglitazone-treated group (data not shown). Since KK-Ay mice ate around 7 g /mouse/day of the chow in the experiment, each mouse was estimated to ingest 17.5–52.5 mg/day of pioglitazone.
injection, while in KK-Ay mice, it was 67 ± 1.1 ng/3000 glomeruli. KK-Ay mice (Fig. 1B) and ICR mice (Fig. 1C) had no histological alterations such as the thickening of capillary walls and expansion of the mesangial matrix in the glomeruli at 7 weeks of age. Levels of serum creatinine and blood urea nitrogen were in the normal range in KK-Ay mice (data not shown). 3.1.1. Effect of long-term treatment with voglibose on diabetic parameters (Fig. 2A, B, C) KK-Ay mice were given mouse-chow containing voglibose at 0.001% or 0.005% for 2 weeks. To clarify the effect of voglibose on diabetic syndrome, blood was drawn for testing. The control KK-Ay mice were found to have 359.4 mg/dl of glucose, 8.3% HbA1C, and 70.8% glycated albumin 2 weeks after the administration of vehicle. The treatment of KK-Ay mice with voglibose suppressed the rise in blood glucose levels, namely a 39% decrease in the 0.001% voglibose-treated group and a 65% decrease in the 0.005% voglibose-treated group as compared with the control KK-Ay group. HbA1C was significantly lower in voglibose-treated KK-Ay mice than vehicle-treated KK-Ay mice (40% and 54% decrease). Voglibose inhibited significantly the increase in glycated albumin in a dose-dependent manner as compared with levels in the vehicle-treated KK-Ay group (43% and 64% decrease). There were no histological alterations in the glomeruli of KK-Ay mice of the control group or voglibose-treated group. Since KK-Ay mice ate about 6 g/mouse/day of the chow in the experiment, each mouse was estimated to ingest 2–6 mg/day of voglibose. 3.1.2. Effect of long-term treatment with voglibose on deposition of aggregated bovine serum albumin in glomeruli (Fig. 2D) KK-Ay mice were given mouse-chow containing 0.001% or 0.005% voglibose for 2 weeks, followed by an i.v. injection of aggregated
Fig. 2. Effect of long-term treatment with voglibose on plasma glucose (A), hemoglobin A1c (HbA1c) (B), and glycated albumin (C) levels, and the deposition of aggregated bovine serum albumin in glomeruli (D) in KK-Ay mice. KK-Ay mice (n = 8) were given mouse-chow containing voglibose at 0.001% or 0.005% for 2 weeks. Blood samples were obtained without fasting (A, B and C). Different groups of KK-Ay mice (n = 4) were treated with voglibose (0.001% or 0.005%) for 2 weeks and were then injected with aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital 3 h after the injection of aggregated bovine serum albumin (D). The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. White columns indicate the control group. Gray columns indicate the voglibose-treated group. Values are means ± S.E.M. ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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1 unit of insulin or vehicle followed by the immediate injection of aggregated bovine serum albumin. There was no significant difference regarding glomerular levels of the aggregated protein between the insulin-treated group and the control group 1 h after injection (Fig. 4B). Insulin-treated KK-Ay mice however had significantly less aggregated bovine serum albumin in their glomeruli than did vehicle-treated KK-Ay mice (431 ng/3000 glomeruli and 615 ng/3000 glomeruli) 3 h after its injection (Fig. 4C). 3.3. Production of advanced glycation endproducts and glycated albumin in vitro (Fig. 5A,B,C)
Fig. 3. Effect of long-term treatment with pioglitazone on plasma glucose (A) and hemoglobin A1c (HbA1c) (B) levels, and the deposition of aggregated bovine serum albumin in glomeruli (C) in KK-Ay mice. KK-Ay mice (n = 8) were given mouse-chow containing pioglitazone at 0.01% or 0.03% for 4 weeks. Blood samples were obtained without fasting. Different groups of KK-Ay mice (n = 4) were treated with pioglitazone (0.01% or 0.03%) for 4 weeks and were then injected with aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital at 3 h after the injection of aggregated bovine serum albumin. The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. Circles indicate the control group. Triangles and squares indicate the 0.01% pioglitazone and 0.03% pioglitazone groups, respectively. White columns indicate the control group. Gray columns indicate the pioglitazone group. Values are means ± S.E.M. ⁎⁎P b 0.01 and ⁎ b 0.05 indicate statistically significant differences from the control group.
Bhatwadekar and Ghole (2005) demonstrated that glycation of albumin was accelerated by L-lysine and higher temperature (50 °C). We determined the amounts of advanced glycation endproducts and glycated albumin that appeared in aggregated bovine serum albumin using their procedure. The fluorescence intensity of advanced glycation endproducts was increased 2.5-fold and 3.5-fold in the 200 mg/dl and 400 mg/dl glucose groups with L-lysine, respectively, compared to those without L-lysine (Fig. 5A). When the mixture of aggregated bovine serum albumin, 600 mg/dl glucose, and L-lysine was incubated, 6 times more advanced glycation endproducts were produced than with bovine serum albumin alone, while 1.5-fold more glycated albumin appeared with bovine serum albumin than with aggregated bovine serum albumin.(Fig. 5B,C) 3.4. Effect of acute hyperglycemia on generation of advanced glycation endproducts and deposition of aggregated bovine serum albumin in glomeruli (Fig. 6A,B,C) Because insulin-treatment caused a decrease in blood glucose levels and the deposition of aggregated bovine serum albumin in KK-Ay mice,
3.1.4. Effect of long-term treatment with pioglitazone on deposition of aggregated bovine serum albumin in glomeruli (Fig. 3C) KK-Ay mice were given mouse-chow containing 0.01% or 0.03% pioglitazone for 4 weeks. The animals were then injected i.v. with aggregated bovine serum albumin, and the kidneys were isolated 3 h later. As shown in Fig. 3C, the vehicle-treated KK-Ay mice had 178 ng/ 3000 glomeruli of aggregated bovine serum albumin after 3 h. KK-Ay mice given 0.03% pioglitazone had significantly less albumin in their glomeruli than the vehicle-treated KK-Ay mice (132 ng/3000 glomeruli, 26% decrease), while 0.01% pioglitazone-treatment failed to decrease the amount of aggregated bovine serum albumin in the glomeruli. 3.2. Effect of short-term insulin-treatment on blood glucose levels and deposition of aggregated bovine serum albumin in glomeruli (Fig. 4A,B,C) We demonstrated that less aggregated protein was deposited in the glomeruli of KK-Ay mice treated with voglibose or pioglitazone than in the glomeruli of vehicle-treated KK-Ay mice. Additional experiments were done to further clarify that hyperglycemia is associated with the deposition of aggregated bovine serum albumin in diabetic glomeruli. KK-Ay mice were injected with 1 unit of insulin, and blood samples were obtained (Fig. 4A). The injection of 1 unit of insulin decreased blood glucose levels from 408 mg/dl to 182 mg/dl after 20 min while in vehicle-treated KK-Ay mice, the level was 458 mg/dl after 20 min. The blood glucose levels were still lower 200 min after the injection of insulin than the glucose levels in the vehicle-treated KK-Ay mice (Fig. 4A). KK-Ay mice were injected with
Fig. 4. Effect of short-term treatment with insulin on blood glucose levels and deposition of aggregated bovine serum albumin in glomeruli of KK-Ay mice. KK-Ay mice were injected s.c. with 1 unit of insulin, and blood was drawn to determine blood glucose levels (A). KK-Ay mice were injected s.c. with 1 unit of insulin before the injection of aggregated bovine serum albumin. The kidneys were obtained under anesthesia with sodium pentobarbital at 1 h (B) and 3 h (C) after the injection. The glomeruli were isolated and the amount of aggregated bovine serum albumin was measured by ELISA. White columns indicate the control group. Gray columns indicate the insulin group. Values are means ± S.E.M. (n = 4). ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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Fig. 5. Production of advanced glycation endproducts (AGEs) and glycated albumin (GA) with aggregated bovine serum albumin (a-BSA) in vitro. (A) The mixture of aggregated bovine serum albumin and glucose was incubated with or without L-lysine. Values are means ± S.E.M. (n = 5). ⁎⁎P b 0.01 indicates statistically significant differences from the fluorescence intensity of the 200 mg/dl glucose group and ##P b 0.01from the fluorescence intensity without L-lysine. (B) The mixture of glucose, proteins (bovine serum albumin or aggregated bovine serum albumin), and L-lysine was incubated. Levels of advanced glycation endproducts were measured by ELISA. Results are indicated as fold values of advanced glycation endproducts produced with bovine serum albumin alone. (C) Glycated albumin is indicated as a percentage of a standard glycated albumin. White columns indicate the control group, gray columns indicate the bovine serum albumin group, and striped columns the aggregated bovine serum albumin group. Values are means ± S.E.M. (n = 4). ⁎⁎P b 0.01 indicates statistically significant differences from the control group and ##P b 0.01from the bovine serum albumin.
we performed experiments to clarify whether acute hyperglycemia induces an increase in the glomerular deposition of aggregated protein and in levels of advanced glycation endproducts in the circulation. ICR mice were administered glucose solution both p.o. and i.p. several times. Blood glucose levels rose from 133 mg/dl to 521 mg/dl at 1 h, and were 640 mg/dl at 4 h after the first administration of the solution (Fig. 6A). Mice were injected with aggregated bovine serum albumin twice, as shown in Fig. 5A. Blood glucose levels were slightly decreased 1 h later (426 mg/dl) although not significantly. After the second injection of aggregated bovine serum albumin, blood glucose levels began to drop and had returned to levels similar to those in the control mice 4 h later (136 mg/dl). Levels of advanced glycation endproducts were measured in plasma obtained 4 h after the injection of aggregated bovine serum albumin. Levels of plasma advanced glycation endproducts were 1.5 times higher in the acute hyperglycemic ICR mice than in the control ICR mice (Fig. 6B). Moreover, as expected, 29% more aggregated bovine serum albumin was detected in the acute hyperglycemic ICR mice than in the control ICR mice (Fig. 6C; 36 ng/104 glomeruli and 28 ng/104 glomeruli).
in KK-Ay mice, and as a result the diabetic glomeruli could still produce additional prostaglandin E2 in response to aggregated protein. De novo prostaglandin E2 then would accelerate the
4. Discussion Previously, we demonstrated that a large amount of exogenous aggregated protein, aggregated bovine serum albumin, accumulated in the glomeruli of diabetic mice as compared with control normal mice (Hirasawa et al., 2006; Nagamatsu et al., 2005). The underlying cause of this phenomenon has yet to be determined. In the present study, we addressed the issue of whether hyperglycemia is associated with the increased deposition of aggregated protein in diabetic glomeruli through analysis of the effect on KK-Ay mice and ICR mice of pharmacological manipulations. We demonstrated that less aggregated protein was deposited in the glomeruli of KK-Ay mice in which the elevation of blood glucose levels was suppressed by long-termtreatment with voglibose or pioglitazone. While prostaglandin E2 plays a critical role in the degradation of aggregated protein in glomeruli (Nagamatsu et al., 2001), diabetic glomeruli showed no increase in the production of prostaglandin E2 in response to aggregated protein in mice with streptozotocin-induced diabetes (Hirasawa et al., 2006). Therefore, it is likely that long-term treatment with voglibose or pioglitazone retarded the development of diabetes
Fig. 6. Effect of acute hyperglycemia on the generation of advanced glycation endproducts (AGEs) in the circulation and glomerular deposition of aggregated bovine serum albumin (a-BSA). D-glucose was dissolved in distilled water at 330 mg/ml. ICR mice were administered glucose solution (3.3 mg/kg body weight) or vehicle. ICR mice were injected with aggregated bovine serum albumin (a-BSA). Blood samples were obtained via an orbital plexus vein. (A) The figure indicates time course of blood glucose. Squares indicate vehicle-treated mice (control), triangles glucose-treated mice, and circles glucose and aggregated bovine serum albumin-treated mice. (B) The figure indicates the effect of hyperglycemia on the generation of advanced glycation endproducts. (C) The figure indicates the effect of hyperglycemia on the deposition of glomeruli. The glomeruli were isolated 4 h after the first injection of aggregated bovine serum albumin. Levels of plasma advanced glycation endproducts were measured 4 h after the first injection of aggregated bovine serum albumin. White columns indicate the control group. Gray columns indicate the hyperglycemia group. Values are means ± S.E. M. (n = 8). ⁎⁎P b 0.01 indicates statistically significant differences from the control group.
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degradation of aggregated protein in diabetic glomeruli. Therefore, we performed an experiment in which a single injection of insulin was given to KK-Ay mice, to avoid the effect of long-term treatment on the kidney. The administration of insulin resulted in lower blood glucose levels and less exogenous aggregated protein in the glomeruli of KK-Ay mice. It seems unlikely that the rate of degradation of aggregated protein returned to the original level in diabetic glomeruli in such a short time after just one injection of insulin. Albumin, immunoglobulin, and advanced glycation endproducts are detected in the glomeruli in experimental models of diabetes (Axe et al., 1981; Figarola et al., 2003; Nakamura et al., 1997). Both glycated albumin and immunoglobulin are detected in the blood of diabetic patients with chronic renal disease (Chujo et al., 2006; Danze et al., 1987). It has also been demonstrated that KK-Ay mice accumulate advanced glycation endproducts, such as CML, in the glomeruli at 20 weeks of age (Ito et al., 2006; Takemoto et al., 2002). The next issue is whether the glycation of aggregated protein and advanced glycation endproducts are generated in the circulation within several hours after the injection of aggregated protein because bovine serum albumin needs to be incubated with high concentrations of glucose at 37 °C for 60 days to produce advanced glycation endproducts in vitro (Gallicchio et al., 2006). Bhatwadekar and Ghole (2005) demonstrated that nonenzymatic glycation of bovine serum albumin and the production of advanced glycation endproducts occurred in a mixture of bovine serum albumin and glucose with L-lysine after a 4 day incubation at 50 °C. In preliminary experiments, we confirmed an increase in the production of advanced glycation endproducts and glycated albumin after a 48 h incubation of aggregated bovine serum albumin as well as serum albumin according to their procedure. Furthermore, in the present study we demonstrated that aggregated serum albumin produced more advanced glycation endproducts than non-aggregated serum albumin (Fig. 5). We then attempted to clarify whether advanced glycation endproducts are generated in blood, and whether aggregated protein is deposited in greater amounts in the glomeruli of short-term hyperglycemic ICR mice. We used short-term hyperglycemia to avoid the effects of long-term hyperglycemia on kidney function. Elevated levels of advanced glycation endproducts and diminished levels of blood glucose were demonstrated in plasma after the injection of aggregated protein. Insulin plays a critical role in the lowering of blood glucose concentrations after the injection of aggregated protein because plasma insulin levels rose with the injection of aggregated protein in KK-Ay mice (Nagamatsu et al., 2005). We consider that, in addition to the effect of insulin, some blood glucose might be consumed by the generation of advanced glycation endproducts and the glycation of aggregated protein. It is worth noting that we detected more aggregated protein in the glomeruli of short-term hyperglycemic ICR mice than in the glomeruli of control mice. Therefore, it seems that the deposition of aggregated protein in glomeruli is closely connected with hyperglycemia. There are many lines of evidence that mesangial cells express scavenger receptors on their cell membrane (Brizzi et al., 2004; Skolnik et al., 1991). Scavenger receptors recognize a broad array of ligands and mediate their internalization for decomposition. Mesangial cells internalize glycated bovine albumin via advanced glycation endproduct receptor 1 (Lu et al., 2004). Yamamoto et al. (2001) demonstrated that mice overexpressing this receptor developed renal dysfunction and glomerulosclerosis when the mice were made diabetic by crossbreeding with another mouse deficient in the islet production of insulin. Taken together, it is speculated that aggregated bovine serum albumin is glycated in the circulation and forms advanced glycation endproducts, and then these products bind to scavenger receptors in glomeruli and are ingested (Tanji et al., 2000). In conclusion, we demonstrated that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli early on in diabetic mice.
References Axe, S.R., Katz, S.M., Lavine, R., 1981. Immunoglobulin deposition in the microvasculature of the streptozotocin-induced diabetic rat. Lab. Invest. 45, 229–233. Bhatwadekar, A.D., Ghole, V.S., 2005. Rapid method for the preparation of an AGE-BSA standard calibrator using thermal glycation. J. Clin. Lab. Anal. 19, 11–15. Bierhaus, A., Hofmann, M.A., Ziegler, R., Nawroth, P.P., 1998. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. Cardiovasc. Res. 37, 586–600. Brizzi, M.F., Dentelli, P., Rosso, A., Calvi, C., Gambino, R., Cassader, M., Salvidio, G., Deferrari, G., Camussi, G., Pegoraro, L., Pagano, G., Cavallo-Perin, P., 2004. RAGE- and TGF-beta receptor-mediated signals converge on STAT5 and p21waf to control cellcycle progression of mesangial cells: a possible role in the development and progression of diabetic nephropathy. FASEB J. 18, 1249–1251. Chujo, K., Shima, K., Tada, H., Oohashi, T., Minakuchi, J., Kawashima, S., 2006. Indicators for blood glucose control in diabetics with end-stage chronic renal disease: GHb vs. glycated albumin (GA). J. Med. Investig. 53, 223–228. Danze, P.M., Tarjoman, A., Rousseaux, J., Fossati, P., Dautrevaux, M., 1987. Evidence for an increased glycation of IgG in diabetic patients. Clin. Chim. Acta 166, 143–153. Figarola, J.L., Scott, S., Loera, S., Tessler, C., Chu, P., Weiss, L., Hardy, J., Rahbar, S., 2003. LR90 a new advanced glycation endproduct inhibitor prevents progression of diabetic nephropathy in streptozotocin-diabetic rats. Diabetologia 46, 1140–1152. Gallicchio, M.A., McRobert, E.A., Tikoo, A., Cooper, M.E., Bach, L.A., 2006. Advanced glycation end products inhibit tubulogenesis and migration of kidney epithelial cells in an ezrin-dependent manner. J. Am. Soc. Nephrol. 17, 414–421. Herz, M., Johns, D., Reviriego, J., Grossman, L.D., Godin, C., Duran, S., Hawkins, F., Lochnan, H., Escobar-Jimenez, F., Hardin, P.A., Konkoy, C.S., Tan, M.H., 2003. A randomized, double-blind, placebo-controlled, clinical trial of the effects of pioglitazone on glycemic control and dyslipidemia in oral antihyperglycemic medication-naive patients with type 2 diabetes mellitus. Clin. Ther. 25, 1074–1095. Hirasawa, Y., Muramatsu, A., Suzuki, Y., Nagamatsu, T., 2006. Insufficient expression of cyclooxygenase-2 protein is associated with retarded degradation of aggregated protein in diabetic glomeruli. J. Pharmacol. Sci. 102, 173–181. Ishida, H., Takizawa, M., Ozawa, S., Nakamichi, Y., Yamaguchi, S., Katsuta, H., Tanaka, T., Maruyama, M., Katahira, H., Yoshimoto, K., Itagaki, E., Nagamatsu, S., 2004. Pioglitazone improves insulin secretory capacity and prevents the loss of betacell mass in obese diabetic db/db mice: possible protection of beta cells from oxidative stress. Metabolism 53, 488–494. Ishizaki, M., Masuda, Y., Fukuda, Y., Yamanaka, N., Masugi, Y., Shichinohe, K., Nakama, K., 1987. Renal lesions in a strain of spontaneously diabetic WBN/Kob rats. Acta Diabetol. Lat. 24, 27–35. Ito, T., Tanimoto, M., Yamada, K., Kaneko, S., Matsumoto, M., Obayashi, K., Hagiwara, S., Murakoshi, M., Aoki, T., Wakabayashi, M., Gohda, T., Funabiki, K., Maeda, K., Horikoshi, S., Tomino, Y., 2006. Glomerular changes in the KK-Ay/Ta mouse: a possible model for human type 2 diabetic nephropathy. Nephrology (Carlton) 11, 29–35. Koyama, M., Wada, R., Mizukami, H., Sakuraba, H., Odaka, H., Ikeda, H., Yagihashi, S., 2000. Inhibition of progressive reduction of islet beta-cell mass in spontaneously diabetic Goto-Kakizaki rats by alpha-glucosidase inhibitor. Metabolism 49, 347–352. Lu, C., He, J.C., Cai, W., Liu, H., Zhu, L., Vlassara, H., 2004. Advanced glycation endproduct (AGE) receptor 1 is a negative regulator of the inflammatory response to AGE in mesangial cells. Proc. Natl. Acad. Sci. U. S. A. 101, 11767–11772. Makita, Z., Bucala, R., Rayfield, E.J., Friedman, E.A., Kaufman, A.M., Korbet, S.M., Barth, R.H., Winston, J.A., Fuh, H., Manogue, K.R., et al.,1994. Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure. Lancet 343, 1519–1522. Mitsuhashi, T., Nakayama, H., Itoh, T., Kuwajima, S., Aoki, S., Atsumi, T., Koike, T., 1993. Immunochemical detection of advanced glycation end products in renal cortex from STZ-induced diabetic rat. Diabetes 42, 826–832. Nagamatsu, T., Hirasawa, Y., Matsui, Y., Ohtsu, S., Yamane, K., Toyoshi, T., Kyuki, K., Suzuki, Y., 2005. Increase in the deposition of aggregated protein in the glomeruli of spontaneously diabetic mice. J. Pharmacol. Sci. 99, 287–293. Nagamatsu, T., Nagao, T., Koseki, J., Sugiura, M., Nishiyama, T., Suzuki, Y., 2001. Involvement of prostaglandin E2 in clearance of aggregated protein via protein kinase A in glomeruli. Jpn. J. Pharmacol. 85, 139–145. Nakamura, S., Makita, Z., Ishikawa, S., Yasumura, K., Fujii, W., Yanagisawa, K., Kawata, T., Koike, T., 1997. Progression of nephropathy in spontaneous diabetic rats is prevented by OPB-9195, a novel inhibitor of advanced glycation. Diabetes 46, 895–899. Okazaki, M., Saito, Y., Udaka, Y., Maruyama, M., Murakami, H., Ota, S., Kikuchi, T., Oguchi, K., 2002. Diabetic nephropathy in KK and KK-Ay mice. Exp. Anim. 51, 191–196. Rahbar, S., Natarajan, R., Yerneni, K., Scott, S., Gonzales, N., Nadler, J.L., 2000. Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation. Clin. Chim. Acta 301, 65–77. Satoh, N., Shimatsu, A., Yamada, K., Aizawa-Abe, M., Suganami, T., Kuzuya, H., Ogawa, Y., 2006. An alpha-glucosidase inhibitor, voglibose, reduces oxidative stress markers and soluble intercellular adhesion molecule 1 in obese type 2 diabetic patients. Metabolism 55, 786–793. Schalkwijk, C.G., Ligtvoet, N., Twaalfhoven, H., Jager, A., Blaauwgeers, H.G., Schlingemann, R.O., Tarnow, L., Parving, H.H., Stehouwer, C.D., van Hinsbergh, V.W., 1999. Amadori albumin in type 1 diabetic patients: correlation with markers of endothelial function, association with diabetic nephropathy, and localization in retinal capillaries. Diabetes 48, 2446–2453. Skolnik, E.Y., Yang, Z., Makita, Z., Radoff, S., Kirstein, M., Vlassara, H., 1991. Human and rat mesangial cell receptors for glucose-modified proteins: potential role in kidney tissue remodelling and diabetic nephropathy. J. Exp. Med. 174, 931–939.
Y. Hirasawa et al. / European Journal of Pharmacology 601 (2008) 129–135 Suzuki, D., 1994. Measurement of the extracellular matrix in glomeruli from patients with diabetic nephropathy using an automatic image analyzer. Nippon Jinzo Gakkai Shi 36, 1209–1215. Takemoto, M., Asker, N., Gerhardt, H., Lundkvist, A., Johansson, B.R., Saito, Y., Betsholtz, C., 2002. A new method for large scale isolation of kidney glomeruli from mice. Am. J. Pathol. 161, 799–805. Takeuchi, M., Bucala, R., Suzuki, T., Ohkubo, T., Yamazaki, M., Koike, T., Kameda, Y., Makita, Z., 2000. Neurotoxicity of advanced glycation end-products for cultured cortical neurons. J. Neuropathol. Exp. Neurol. 59, 1094–1105. Tanaka, Y., Uchino, H., Shimizu, T., Yoshii, H., Niwa, M., Ohmura, C., Mitsuhashi, N., Onuma, T., Kawamori, R., 1999. Effect of metformin on advanced glycation endproduct formation and peripheral nerve function in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 376, 17–22. Tanji, N., Markowitz, G.S., Fu, C., Kislinger, T., Taguchi, A., Pischetsrieder, M., Stern, D., Schmidt, A.M., D'Agati, V.D., 2000. Expression of advanced glycation end products
135
and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease. J. Am. Soc. Nephrol. 11, 1656–1666. Vlassara, H., Striker, L.J., Teichberg, S., Fuh, H., Li, Y.M., Steffes, M., 1994. Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc. Natl. Acad. Sci. U. S. A. 91, 11704–11708. Yamagishi, S., Yonekura, H., Yamamoto, Y., Katsuno, K., Sato, F., Mita, I., Ooka, H., Satozawa, N., Kawakami, T., Nomura, M., Yamamoto, H., 1997. Advanced glycation end products-driven angiogenesis in vitro. Induction of the growth and tube formation of human microvascular endothelial cells through autocrine vascular endothelial growth factor. J. Biol. Chem. 272, 8723–8730. Yamamoto, Y., Kato, I., Doi, I., Yonekura, H., Ohashi, S., Takeuchi, M., Watanabe, T., Yamagishi, S., Sakurai, S., Takasawa, S., Okamoto, H., Yamamoto, H., 2001. Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice. J. Clin. Invest. 108, 261–268.