Background

Gliclazide is a sulphonylurea antidiabetic drug with anti-oxidant effect due to its azabicyclo-octyl ring. We hypothesized that gliclazide may have a beneficial effect on diabetic nephropathy via radical scavenging.

Methods

Streptozotocin-induced diabetic rats fed a 4% cholesterol diet [high cholesterol-diabetes mellitus (HC-DM)] (N = 12) were treated with gliclazide (HC-DM + gliclazide) (N = 12) or glibenclamide (HC-DM + glibenclamide) (N = 12) after 2 weeks of the diabetes induction, and normal rat fed with 4% cholesterol served as control [high cholesterol-control (HC-control)] (N = 12). Renal expression of endothelial nitric oxide synthase (eNOS) and intracellular adhesion molecule-1 (ICAM-1), oxidative stress production via nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase and antioxidant enzyme manganese superoxide dismutase (MnSOD) were evaluated at 4 weeks and renal damage was examined at 8 weeks.

Results

HC-DM showed significant increase in renal NAD(P)H oxidase and reduction in MnSOD with a significant increase in urinary lipid peroxidation products and H₂O₂ excretion compared to HC-control. Gliclazide treatment, but not glibenclamide, significantly reduced the oxidative products and NAD(P)H oxidase. There was no difference in renal eNOS expression between HC-DM and HC-control rats, and only gliclazide treatment enhanced eNOS expression. Renal damage evaluated by increased glomerular macrophage migration via enhanced ICAM-1 expression, mesangial matrix expansion, and albuminuria was significantly increased in HC-DM, and they were ameliorated by gliclazide but not by glibenclamide.

Conclusion

Gliclazide reduced oxidative stress in diabetic rats fed a high cholesterol diet with reduction of renal NAD(P)H oxidase expression, enhanced MnSOD and eNOS expression, and had a beneficial effect on glomerular macrophage infiltration and mesangial expansion.

METHODS

Protocol 1

Female Sprague-Dawley rats weighing 180 to 240 g (Charles River Laboratories, Shizuoka, Japan) were housed in a temperature-and humidity-controlled room with a 12-hour light/dark cycle and were fed standard laboratory animal chow and had free access to tap water. Diabetes mellitus (DM) was induced by a single tail vein injection of streptozotocin (STZ), 60 mg/kg body weight; N = 10) (Sigma Chemical Co., St. Louis, MO, USA) diluted in citrate buffer, pH 4.5. Control rats (N = 10) were injected with an equal volume of citrate buffer. Another set of DM and control rats (12 animals in each group) were fed 4% cholesterol diet with 1% cholic acid [high cholesterol-diabetes mellitus (HC-DM) and high cholesterol-control (HC-control)] to increase macrophage infiltration in the glomeruli. After 3 days, the induction of diabetes was confirmed by measurement of blood glucose concentration.

Four weeks after STZ injection 24-hour urine and blood samples were collected. The animals were anesthetized with pentobarbital (50 mg/kg body weight). Abdominal aorta was cannulated, the kidneys were retrograde perfused with ice-cold phosphate buffered saline (PBS) and the right kidney was taken and immediately frozen for Western blotting and the left kidney was perfused with periodate-lysine-paraformaldehyde (PLP) solution and embedded in wax for immunohistochemical study.

Protocol 2

Two weeks after STZ injection, HC-DM rats were randomly divided into three groups matched for body weight and blood glucose: (1) diabetic untreated (HC-DM) (N = 12); (2) gliclazide-treated (HC-DM + gliclazide) (N = 12); and (3) glibenclamide-treated (HC-DM + glibenclamide) (N = 12). Gliclazide (150 mg/kg/day) and glibenclamide (8.6 mg/kg/day) were donated by Dainippon Pharmaceutical Co. Ltd., Osaka, Japan and were given in the diet. Female Sprague-Dawley rats fed 4% cholesterol diet with 1% cholic acid served as control (HC-control) (N = 12). After 2 weeks of treatment and 4 weeks of diabetes induction, 24-hour urine and blood were collected and rats were sacrificed as described above.

Another set of five rats in each of the four groups underwent the same procedures as described above and were treated until 8 weeks after STZ injection. They were used for evaluation of the effects of treatment on renal damage, including glomerular macrophage migration, mesangial matrix expansion, and albuminuria. All procedures were conducted in conformance with the Guide for Animal Experimentation of the Faculty of Medicine of the University of Tokyo.

Western blotting

As described in detail previously^5,9 kidneys were homogenized on ice with a Teflon-glass tissue homogenizer (Iwaki, Chiba, Japan) in 3 mL of 20 mmol/L Tris and homogenates were centrifuged at 4°C and 12,000 rpm for 20 minutes. The supernatants were diluted in the same volume of sodium dodecyl sulfate (SDS) buffer and samples containing 25 g of protein were applied to 4% to 20% gradient gel (Daiichi Pure Chemicals Co., Tokyo, Japan) and electroblotted to nitrocellulose membranes. The membranes were incubated with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 minutes, following overnight incubation with 15 g/mL of monoclonal antibody for 4-hydroxy-2-nonenal (4-HNE) (JaICA, Shizuoka, Japan), monoclonal antibody for p47phox (Transduction Laboratories, Lexington, KY, USA), monoclonal antibody for manganese SOD (MnSOD) (Transduction Laboratories, Lexington, KY, USA), polyclonal antibody for endothelial nitric oxide synthase (eNOS) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), in a 1:1000 dilution and monoclonal antibody for intracellular adhesion molecule-1 (ICAM-1) (Serotec, Oxford, England) in a 1:100 dilution. After rinsing in TBST, membranes were incubated for 2 hours with a horseradish peroxidase (HRP)-conjugated secondary antibody against mouse IgG (Dako, Glostrup, Denmark) for 4-HNE, p47phox, MnSOD, and ICAM-1, or against rabbit IgG (Dako) for eNOS in a 1:1000 dilution, and rinsed with TBST followed by 0.8 mmol/L diaminobenzidine (DAB) with 0.01% H₂O₂ and 3 mmol/L NiCl₂ for the detection of blots. The density of the bands was analyzed using NIH image analyzer computer program.

Immunohistochemistry and morphologic studies

The tissues were embedded in wax for periodic acid-Schiff staining (PAS) and light microscopic immunohistochemistry. Kidney slices were processed for immunohistochemistry using the labeled streptavidin-biotin method as described previously^5,9. Sections (2 m) were dewaxed, incubated with 3% H₂O₂, blocking serum, and thereafter with a polyclonal antibody against eNOS (Santa Cruz Biotechnology, Inc.) and monoclonal antibody against endothelin-1 (ED-1) (BMA Biomedicals AG, Augst, Switzerland) at 1:100 dilution. The sections were rinsed with TBST and biotinylated secondary antibody against rabbit IgG (Dako) for eNOS, or mouse IgG (Dako) for ED-1 in a 1:400 dilution. After rinsing with TBST, the sections were incubated with HRP-conjugated streptavidin solution (Dako). HRP labeling was detected using a peroxide substrate solution with 0.8 mmol/L DAB and 0.01% H₂O₂. The sections were counterstained with hematoxylin before being examined under a light microscope.

Mesangial matrix expansion was analyzed in all glomeruli of each section as S0 for a normal glomerulus; S1, <25% of glomerular area; S2, 25% to 50%; S3, 50% to 75%; S4, >75%. The degree of mesangial matrix expansion was expressed as: mesangial matrix expansion index =[0 number of S0 + 1 number of S1 + 2 number of S2 + 3 number of S3 + 4 number of S4]/[number of S0 + number of S1 + number of S2 + number of S3 + number of S4]. The number of glomerular macrophage infiltration was calculated as a mean of all glomeruli in each cross-section from each animal.

Measurement of glucose, H₂O₂, lipid peroxidation products, nitrite, creatinine, blood lipids, and albuminuria

All procedures for measurements were described in detail in our previous reports^5,24. Blood glucose was measured by Glutest E II (Kyoto Dai-iti Kagaku, Kyoto, Japan). The H₂O₂ production was measured by oxidation of nonfluorescent 2',7'-dichlorodihydrofluorescin-diacetate (DCFH-DA) to the fluorescent 2',7'-dichlorodihydrofluorescein (DCF). Samples were incubated with 16 L/mL final concentration of DCFH-DA for 20 minutes at 37°C, and highly fluorescent DCF formed in the presence of H₂O₂ was measured in a spectrofluorometer (Hitachi F-2000) (Hitachi, Tokyo, Japan) with wavelength at 485/535 nm. The lipid peroxidation products (LPO) were measured by thiobarbituric acid method. Briefly, after precipitation of proteins, 100 L of urine was incubated with 100 L of 4% sodium dodecyl sulfate (SDS), 400 L of 20% acetic acid at pH 3.5 and 400 L of 0.8% 2-thiobarbituric acid (TBA) (Wako Pure Chemical Industries LTD., Osaka, Japan) for 60 minutes at 95°C. The malondialdehyde (MDA) formation was measured with a spectrofluorometer with an excitation/emission wavelength at 515/553 nm. Urinary excretion of nitrite was measured according to the Griess method as described previously²⁴. After precipitation of protein by adding an equal amount of 0.3 N NaOH and 5% ZnSO₄ and centrifugation at 6400 rpm during 20 minutes, supernatants were reacted with Griess solution, and nitrite was measured with a spectrophotometer at 540 nm wavelength. Creatinine in urine and blood was measured by Jaffe method using spectrophotometer. Blood total cholesterol and triglycerides were measured with an autoanalyzer. Urinary albumin was quantified by enzyme-linked immunosorbent assay (ELISA) kit (Panaform Lab., Kumamoto, Japan).

Statistics

All data were expressed as means SEM. The analysis of variance with Bonferroni's post hoc test was used for statistical comparisons in the data with normal distribution, including mesangial matrix expansion index, mean glomerular macrophage infiltration number, densitometry of Western blot, and other biochemical measurements. The data that did not show normal distribution such as urinary albumin concentration were evaluated with nonparametric analysis with Mann-Whitney test or logarithmically calculated before being submitted to analysis of variance (ANOVA). P values less than 0.05 were required for statistical significance.

RESULTS

Effect of high cholesterol diet in diabetes

We confirmed our previous publication⁵ that diabetic rats fed with a normal diet (DM) have an increased expression of NAD(P)H oxidase isoform, p47phox, with an increased formation of superoxide anion and an increase in eNOS expression in the kidney at 4 weeks Table 1. We evaluated the effect of a high cholesterol diet on NAD(P)H oxidase isoform p47phox and eNOS in control rats fed with a high cholesterol diet (HC-control) and diabetic rats fed with a high cholesterol diet (HC-DM) and compared to animals fed with a normal diet.

Table 1 - Data of experimental animals fed a normal diet or high cholesterol diet at 4 weeks of diabetes induction.

Full table

We found that renal NAD(P)H oxidase p47phox was significantly increased in high cholesterol diet compared to normal diet in diabetic rats but not in control rats Table 1. Following p47phox increase, urinary LPO excretion was markedly increased in HC-DM compared to DM. This effect was less prominent in HC-control compared to control. Enhanced renal eNOS expression in DM was decreased in HC-DM to the same level shown in HC-control Table 1. There was a significant increase in glomerular macrophage infiltration in DM compared to control rats, and cholesterol diet addictively increased macrophage migration in both groups however HC-DM showed a more pronounced infiltration than HC-control Table 1. This increased macrophage migration can be explained by the increased renal ICAM-1 expression caused by the reduced eNOS and enhanced oxidative stress in HC-DM rats Table 1.

Effect of gliclazide and glibenclamide in physiologic data and hemodynamics at 4 weeks of diabetes induction

Diabetic rats fed a high cholesterol diet (HC-DM) at 4 weeks showed a significant increase in blood glucose and hemoglobin A_1c (HbA_1c) and a reduction of body weight, but no change in the mean blood pressure compared to HC-control. The creatinine clearance was slightly higher in HC-DM rats but the difference was not significant under fasting condition Table 2. Total cholesterol and triglyceride were significant increased in HC-DM than HC-control Table 2. Treatment with gliclazide and glibenclamide did not change blood glucose level, HbA_1c, total cholesterol, and mean blood pressure compared to HC-DM. Gliclazide-treated animals (HC-DM + gliclazide) showed an increase in creatinine clearance compared to control (HC-control) and a significant decrease in triglyceride levels compared to HC-DM Table 2.

Table 2 - Effect of gliclazide and glibenclamide treatment on physiologic data of high cholesterol–fed diabetic rats at 4 weeks.

Full table

Oxidative stress, NAD(P)H oxidase, and MnSOD

HC-DM rats showed a significant increase in LPO excretion in urine and an increase tendency in kidney 4-HNE production compared to HC-control Table 2. Gliclazide treatment effectively decreased 4-HNE expression in the kidney and LPO production compared to the HC-DM group, while no effect was observed with glibenclamide Table 2, indicating that only gliclazide has a radical scavenging action.

NAD(P)H oxidase has been shown an important source of oxidative stress and the quantitative analysis of its component p47phox protein in the kidney showed a specific band corresponding to a molecular weight of 47 kD by Western blotting. Densitometry of the band showed increased protein amount in HC-DM compared to HC-control rats (0.32 0.02 vs. 0.23 0.02 arbitrary units, P < 0.01) Figure 1. This was decreased by treatment with gliclazide (0.21 0.02, P < 0.001 vs. HC-DM) but not with glibenclamide (0.26 0.02, NS vs. HC-DM) Figure 1. On the other hand, Western blot analysis of the superoxide scavenge system, MnSOD protein in kidney showed a band of 25 kD that was significantly decreased in the HC-DM group compared to HC-control (0.20 0.01 vs. 0.31 0.02 arbitrary units, P < 0.0001) Figure 2, supporting the enhanced oxidative stress in HC-DM. Gliclazide reversed this expression to the control level (0.30 0.01, NS vs. HC-control) Figure 2, but glibenclamide did not increase MnSOD expression (0.19 0.02, P < 0.0001 vs. HC-control) Figure 2. The reduction of oxidative stress by gliclazide may also be explained by the enhanced expression of MnSOD.

Figure 1.

Western blotting analysis of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase isoform p47phox in the kidney. Densitometry of bands at molecular weight of 47 kD showed a significant increase in NAD(P)H oxidase p47phox expression in diabetic rats fed a high cholesterol diet (HC-DM) compared to control rats fed the same diet (HC-control). Treatment with gliclazide (HC-DM + gliclazide) was effective in lowering p47phox expression in the kidney; however, this was not observed in the glibenclamide-treated animals (HC-DM + glibenclamide). ⁺P < 0.01 vs. HC-control; *P < 0.001 vs. HC-DM (N = 10 in each group).

Full figure and legend (69K)

Figure 2.

Western blotting analysis of manganese superoxide dismutase (MnSOD). The densitometry of MnSOD band at 25 kD showed a significant decrease in diabetic rats fed with a high cholesterol diet (HC-DM) compared to control rats fed the same diet (HC-control) at 4 weeks. Only treatment with gliclazide (HC-DM + gliclazide), but not glibenclamide (HC-DM + glibenclamide) reversed MnSOD to the control level. ⁺P < 0.0001 vs. HC-control; *P < 0.0001 vs. HC-DM; ^#P < 0.0001 vs. HC-DM + glibenclamide (N = 10 in each group).

Full figure and legend (76K)

eNOS expression and nitrite production in the kidney

Nitric oxide produced by eNOS counteracts superoxide anion and has an inhibitory action on adhesion molecules in the renal vessels, thus we examined eNOS immunoreactivity in endothelial cells of renal arteries in HC-DM rats. It was comparable to that in HC-control rat, and gliclazide treatment, but not glibenclamide, enhanced eNOS expression in renal vasculature Figure 3. Western blot analysis confirmed no difference in eNOS expression in HC-DM compared to HC-control rats (0.24 0.01 vs. 0.25 0.01 arbitrary units, NS) Figure 4, and increase in eNOS expression with gliclazide treatment (0.29 0.01, P < 0.05 vs. HC-DM) but no effect with glibenclamide (0.22 0.02, NS). These changes of eNOS expression were confirmed by nitrite production in the kidney. HC-DM did not show difference in nitrite production compared to HC-control; however, gliclazide treatment significantly increased nitrite production but this was not observed with glibenclamide Table 2.

Figure 3.

Immunohistochemistry of endothelial nitric oxide synthase (eNOS) in the renal artery. Immunoreactivity for eNOS in the endothelial cells of renal artery showed no difference between control rats fed a high cholesterol diet (HC-control) (A) and diabetic rats fed with a high cholesterol diet (HC-DM) (B). Treatment of diabetic rats with gliclazide (HC-DM + gliclazide) (C) showed increased immunoreactivity for eNOS but no effect with glibenclamide (HC-DM + glibenclamide) (D) (magnification 800).

Full figure and legend (219K)

Figure 4.

Western blotting analysis for endothelial nitric oxide synthase (eNOS) in kidney homogenates. The densitometry of eNOS band at 133 kD showed no statistically significant change between control (HC-control) and diabetic rats (HC-DM) fed a high cholesterol diet at 4 weeks. Treatment with gliclazide (HC-DM + gliclazide) up-regulated eNOS expression. Glibenclamide (HC-DM + glibenclamide) did not have any effect on eNOS expression in the kidney. ⁺P < 0.05 vs. HC-control; *P < 0.05 vs. HC-DM; ^#P < 0.005 vs. HC-DM + glibenclamide (N = 10 in each group).

Full figure and legend (68K)

ICAM-1 expression, macrophage infiltration, and renal damage

The adhesion molecule ICAM-1 was up-regulated in the kidney of diabetic rats compared to control (0.32 0.02 vs. 0.19 0.02 arbitrary units, P < 0.0001) Figure 5. Following the increased protein expression of ICAM-1 in the kidney, glomerular macrophage infiltration detected by immmunostaining with its maker ED-1 was significantly increased in diabetes Figure 6 and the average number of macrophages in each glomerulus was about two times higher in diabetic kidneys compared to control (2.27 0.10 vs. 1.15 0.07, P < 0.0001). Gliclazide treatment significantly reduced ICAM-1 expression in the kidney (0.20 0.01, P < 0.0001 vs. HC-DM) Figure 5 followed by a decrease in macrophage infiltration in the glomeruli (1.85 0.08, P < 0.0001 vs. HC-DM) Figure 6 while glibenclamide treatment showed no effect in ICAM-1 expression (0.29 0.02, NS vs. HC-DM) Figure 5 and macrophage infiltration (2.24 0.08, NS vs. HC-DM) Figure 6 compared to HC-DM rats. Diabetic rats presented markedly increase in urinary albumin excretion, which was significantly reduced with treatment with gliclazide Table 2. Treatment with glibenclamide showed no significant change in urinary albumin excretion Table 2.

Figure 5.

Western blotting analysis for intracellular adhesion molecule-1 (ICAM-1) in kidney. ICAM-1 showed a significant increase in diabetic rats fed a high cholesterol diet (HC-DM) compared to control animals given a high cholesterol diet (HC-control) at 4 weeks, and gliclazide (HC-DM + gliclazide) effectively lowered ICAM-1 expression to control levels. ⁺P < 0.0005; ⁺⁺P < 0.0001 vs. HC-control; *P < 0.0001 vs. HC-DM; ^#P < 0.001 vs. HC-DM + glibenclamide (N = 10 in each group).

Full figure and legend (72K)

Figure 6.

Immunostaining for endothelin-1 (ED-1) in glomeruli. Macrophage infiltration in glomeruli was observed by immunostaining with the macrophage marker ED-1 at 4 weeks in control rats fed a high cholesterol diet (HC-control) (A), diabetic rats fed a high cholesterol diet (HC-DM) (B), HC-DM rats treated with gliclazide (HC-DM + gliclazide) (C), and HC-DM rats treated with glibenclamide (HC-DM + glibenclamide) (D) (magnification 360).

Full figure and legend (302K)

Effect of gliclazide and glibenclamide on renal damage at 8 weeks

At 8 weeks, HC-DM showed significant increase in blood glucose and HbA_1c compared to HC-control. HbA_1c value was not affected by gliclazide treatment; however, glibenclamide-treated group showed a slight decrease compared to nontreated diabetic rats. HC-DM rats showed an increased oxidative stress evaluated by urinary H₂O₂ excretion compared to HC-control, and only gliclazide reduced it significantly Table 3. Nitrite formation in the kidney did not show difference at 8 weeks between HC-DM and HC-control, and treatment with gliclazide significantly increased renal nitrite Table 3.

Table 3 - Effect of gliclazide and glibenclamide treatment on high cholesterol–fed diabetic rats at 8 weeks.

Full table

With the evolution of diabetic nephropathy, HC-DM showed mesangial matrix expansion compared to HC-control and further increase in glomerular macrophage migration Table 3. These renal changes were accompanied by increased urinary albumin excretion in HC-DM compared to HC-control Table 3. Gliclazide treatment ameliorated mesangial matrix expansion and decreased glomerular macrophage infiltration compared to HC-DM, and showed a slight decrease in albuminuria that was statistically not different from HC-control Table 3. Glibenclamide showed no effect on these parameters of renal damage Table 3.

DISCUSSION

In this study we firstly confirmed our previous report⁵ that showed enhanced NAD(P)H and eNOS in the early stage of diabetes with normal diet, and we revealed that high cholesterol diet increased oxidative stress and glomerular macrophage migration in the kidney of normal and diabetic rats by the enhanced NAD(P)H oxidase p47phox expression and reduction of eNOS expression. Secondly we demonstrated a beneficial effect of gliclazide in diabetic nephropathy independent of insulin secretion and glycemic control. This effect of gliclazide is due to scavenge of ROS via its azabicyclo-octyl ring structure, NAD(P)H oxidase suppression and up-regulation of MnSOD, and also due to increased nitric oxide bioavailability with enhanced eNOS leading to ICAM-1 suppression and reducing glomerular macrophage infiltration. These beneficial effects were not observed with the other sulphonylurea glibenclamide.

It has been shown that ROS production is enhanced in diabetic rats via high glucose, de novo synthesis of diacylglycerol, and enhanced protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK) signals and so on^2,25. We have previously demonstrated that NAD(P)H oxidase expression and its products are enhanced in the kidney of diabetic rat⁵. In this study, we showed that the high cholesterol diet further increased renal NAD(P)H oxidase in diabetic rats. Interestingly, MnSOD expression in the kidney was also significantly decreased and this may have contributed to a further enhancement of ROS in HC-DM rat compared to HC-control rat. We showed that only gliclazide, but not glibenclamide, could lower ROS production. The effect of gliclazide may depend on its specific molecular structure with an azabicyclo-octyl ring to scavenge superoxide anion and hydroxyl radicals^17,26. Moreover, gliclazide also significantly inhibited NAD(P)H oxidase and increased the expression of MnSOD in the kidney, contributing to reduce ROS. Further studies are necessary to reveal the mechanism of gliclazide stimulation of MnSOD and inhibition of NAD(P)H oxidase.

The eNOS expression and its product nitric oxide have an important role in the prevention of diabetic nephropathy. The eNOS protein and mRNA levels were increased in aorta of early phase diabetic rats to counterbalance the increased degradation of nitric oxide by the reaction with superoxide²⁷. We have also previously demonstrated that eNOS expression was increased in the kidney of early phase of diabetes in rats accompanied by increased superoxide production and increased nitrosylation of proteins by the reaction of nitric oxide with superoxide⁵. However, eNOS did not increase in HC-DM and this may be explained by the evidence that a high cholesterol diet decreases eNOS protein amount in renal arteries²⁸. Likewise, administration of antioxidants can prevent the fall in eNOS expression in hypercholesterolemia²⁹. We observed that gliclazide, but not glibenclamide, effectively increased eNOS expression and nitric oxide production in the kidney. It was reported that eNOS deficiency and plasma lipid dysfunction favors end-organ damage³⁰ and compounds that specifically up-regulate this enzyme are desirable for organ protection³¹. Thus, enhanced eNOS and increased nitric oxide bioavailability by gliclazide may contribute to renoprotection in the diabetic nephropathy.

The macrophages have been shown to play an important role in the progression of glomerulosclerosis in diabetic nephropathy^21,32,33. The increased infiltration of macrophages follows the increased expression of adhesion molecules. The adhesion molecule, ICAM-1, is up-regulated in the early stage of diabetes and promotes the recruitment of macrophages in diabetic glomeruli³⁴. ICAM-1 is constitutively expressed in endothelial cells, and its expression can be induced on mesangial cells and epithelial cells by glucose, PKC, and intracellular free radicals, angiotensin II, and cytokines via nuclear factor-B (NF-B). On the other hand, nitric oxide down-regulates its expression and impedes the adhesion of inflammatory cells^34,35,36. We have previously shown that eNOS enhancement suppresses renal ICAM-1 expression and decreases glomerular macrophage migration in the nephrotoxic nephritis²⁴. Similarly in this diabetic model, renal eNOS enhancement with increased nitric oxide bioavailability by gliclazide suppressed the ICAM-1 expression and decreased glomerular macrophage migration. This effect of gliclazide on macrophage infiltration via adhesion molecules is consistent with previous reports in the retinal vasculature of diabetic rat and human^37,38. Because the adhesion of phagocytes to glomerular cells can further increase free oxygen radical production and cause renal damage, inhibition of glomerular macrophage infiltration via suppression of ICAM-1 may explain the reduction of albuminuria in the HC-DM rat treated with gliclazide at 4 weeks.

With longer treatment with gliclazide at 8 weeks we found a decreased production of H₂O₂ production and decreased infiltration of macrophage and glomerular matrix expansion in the HC-DM + gliclazide group. Albuminuria was not significantly decreased in the HC-DM + gliclazide group compared to the HC-DM group, but it was not significantly increased compared to HC-control. On the other hand, albuminuria was significantly higher in the HC-DM + glibenclamide group then in HC-control, suggesting that gliclazide is still beneficial on albuminuria than glibenclamide in the longer treatment. Jerums et al³⁹ did not find a significant reduction of albuminuria after 2 years of treatment with gliclazide in humans; however, they could not totally exclude the possibility of gliclazide in preventing the progression to overt diabetic nephropathy. It is possible that the morphologic benefit seen with gliclazide in our study results from its action on radical production. However, it is also possible that this model is not totally transferable to humans. At 8 weeks, HbA_1c was slightly but not significantly lower in HC-DM + gliclazide group and it was significantly reduced in HC-DM + glibenclamide group compared to HC-DM group; thus, the antidiabetic effect of sulfonylureas on diabetic nephropathy cannot be completely excluded in the longer treatment.

CONCLUSION

Among the sulfonylurea antidiabetic drugs, gliclazide has a specific characteristic of scavenging ROS, reducing the expression of NAD(P)H oxidase and enhancing renal expression of MnSOD and eNOS. Increased nitric oxide bioavailability by gliclazide suppressed glomerular macrophage migration via decrease in ICAM-1 expression and ameliorated glomerular matrix expansion and albuminuria.

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Acknowledgments

This work was supported by a grant to Dr. A. Tojo (grant #C2-14571014) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.