Liraglutide alleviates arteriosclerotic lesions and inhibits receptor for advanced glycation end-product expression in diabetic ApoE-/- mice / 中华内分泌代谢杂志

Objective To observe the effect of liraglutide on arteriosclerotic lesions in diabetic ApoE- / - mice via regulating receptor for advanced glycation end-products ( RAGE) expression of aorta. Methods Thirty male ApoE- / - mice (10 weeks) were randomly divided into 3 groups: control group, diabetes group, and diabetes +liraglutide group. All the mice were fed a high-fat diet. Diabetes was induced by 2 intraperitoneal injection of streptozotocin (STZ, 55 mg·kg-1 ·d-1 ). Mice were treated with subcutaneous injection of liraglutide (0. 4 mg· kg-1 ·d-1 ) in diabetes+liraglutide group. After 9 weeks, blood was drawn and the aorta were rapidly procured. The serum levels of advanced glycation end-products ( AGEs), soluble receptor for advanced glycation end-products (sRAGE ), stromal cell-derived factor-1α ( SDF-1α ), as well as total cholesterol and triacylglycerol were measured. Atherosclerotic plaque area was determined by Sudan Ⅳ staining. The expression of RAGE was determined using RT-PCR and western blot, respectively. Results Compared with control group, the serum AGEs, the RAGE protein and mRNA expression, the atherosclerotic lesions were significantly increased in the mice of diabetes group. Compared with diabetes group, the serum AGEs, the RAGE protein and mRNA expression, the atherosclerotic lesions were significantly decreased in the mice of diabetes+liraglutide group. Conclusion Liraglutide is confirmed as to have an anti-atherosclerotic effects and inhibits RAGE expression in diabetic ApoE- / - mice.

Ginseng Total Saponin Improves Podocyte Hyperpermeability Induced by High Glucose and Advanced Glycosylation Endproducts

Early diabetic nephropathy is characterized by glomerular hyperpermeability as a result of impaired glomerular filtration structure caused by hyperglycemia, glycated proteins or irreversible advanced glycosylation endproducts (AGE). To investigate the effect of ginseng total saponin (GTS) on the pathologic changes of podocyte ZO (zonula occludens)-1 protein and podocyte permeability induced by diabetic conditions, we cultured mouse podocytes under: 1) normal glucose (5 mM, = control); 2) high glucose (HG, 30 mM); 3) AGE-added; or 4) HG plus AGE-added conditions and treated with GTS. HG and AGE increased the dextran filtration of monolayered podocytes at early stage (2-8 hr) in permeability assay. In confocal imaging, ZO-1 colocalized with actin filaments and beta-catenin at cell contact areas, forming intercellular filtration gaps. However, these diabetic conditions suppressed ZO-1 immunostainings and disrupted the linearity of ZO-1. In Western blotting, diabetic conditions also decreased cellular ZO-1 protein levels at 6 hr and 24 hr. GTS improved such quantitative and qualitative changes. These findings imply that HG and AGE have an influence on the redistribution and amount of ZO-1 protein of podocytes thereby causing hyperpermeability at early stage, which can be reversed by GTS.

Beneficial effects of intensive therapy on arterial intima-media thickness and its risk factors in type 2 diabetic patients / 中华内分泌代谢杂志

Objective To analyze the changes of the intima-media thickness(IMT)of carotid and femoral arteries, serum advanced glycosylation end-products(AGEs),and AGEs soluble receptor(sRAGE)after intensively controlling blood glucose, blood pressure, and lipid. Methods One hundred and thirty-two type 2 diabetic patients were divided into 3 groups and followed for 5 years: 20 patients were treated with intensive control of blood glucose and blood pressure, 80 patients with intensive control of blood glucose, blood pressure, and lipid; and 32 patients with conventional therapy. AGEs, sRAGE, and IMT of carotid and femoral arteries were measured and compared among different groups. Results The IMT of carotid and femoral arteries and serum level of AGEs were significantly decreased after intensive treatment. The ratio of sRAGE and HbA1C(sRAGE/HbA1C)were negatively correlated with the mean of HbA1Cin the past five years(r=-0.417, P＜0.001)and the fluctuation of HbA1C(r=-0.309,P＜0.001). Multinomial regression analysis showed that AGEs were the important risk factors of IMT of femoral artery(β=0.152,P=0.068). Conclusion Intensive treatment is significant in controlling the growing IMT of carotid and femoral arteries, while decreasing serum level of AGEs.

Diabetic Nephropathy in Childhood and Adolescence (II); Pathology and Pathophysiology

Diabetic nephropathy is a major cause of chronic renal failure in developing countries, and the prevalence rate has markedly increased during the past decade. Diabetic nephropathy shows various specific histological changes not only in the glomeruli but also in the tubulointerstitial region. In the early stage, the effacement of podocyte foot processes and thickened glomerular basement membrane (GBM) is noticed even at the stage of microalbuminuria. Nodular, diffuse, and exudative lesions, so-called diabetic glomerulosclerosis, are well known as glomerular lesions. Interstitial lesions also exhibit fibrosis, edema, and thickened tubular basement membrane. Diabetic nephropathy is considered to be multifactorial in origin with increasing evidence that one of the major pathways involved in the development and progression of diabetic nephropathy as a result of hyperglycemia. Hyperglycemia induces renal damage directly or through hemodynamic alterations, such as, glomerular hyperfiltration, shear stress, and microalbuminuria. Chronic hyperglycemia also induces nonhemodynamic dysregulations, such as, increased production of advanced glycosylation endproducts, oxidative stress, activation of signal pathway, and subsequent various cytokines. Those pathogenic mechanisms resulted in extracellular matrix deposition including mesangial expansion and GBM thickening, glomerular hypertrophy, inflammation, and proteinuria. In this review, recent opinions on the histopathologic changes and pathophysiologic mechanisms leading to initiation and progression of diabetic nephropathy will be introduced.

Effects of High Glucose and Advanced Glycosylation Endproducts(AGE) on the in vitro Permeability Model

PURPOSE: We describe the changes of rat glomerular epithelial cells when exposed to high levels of glucose and advanced glycosylation endproducts(AGE) in the in vitro diabetic condition. We expect morphological alteration of glomerular epithelial cells and permeability changes experimentally and we may correlate the results with a mechanism of proteinuria in DM. METHODS: We made 0.2 M glucose-6-phsphate solution mixed with PBS(pH 7.4) containing 50 mg/mL BSA and protease inhibitor for preparation of AGE. As control, we used BSA. We manufactured and symbolized five culture dishes as follows; B5 - normal glucose(5 mM) + BSA, B30 - high glucose(30 mM) + BSA, A5 - normal glucose(5 mM) + AGE, A30 - high glucose(30 mM) + AGE, A/B 25 - normal glucose(5 mM) + 25 mM of mannitol(osmotic control). After the incubation period of both two days and seven days, we measured the amount of heparan sulfate proteoglycan(HSPG) in each dish by ELISA and compared them with the B5 dish at 2nd and 7th incubation days. We observed the morphological changes of epithelial cells in each culture dish using scanning electron microscopy(SEM). We tried the permeability assay of glomerular epithelial cells using cellulose semi-permeable membrane measuring the amount of filtered BSA through the apical chamber for 2 hours by sandwich ELISA. RESULTS: On the 2nd incubation day, there was no significant difference in the amount of HSPG between the 5 culture dishes. But on the 7th incubation day, the amount of HSPG increased by 10% compared with the B5 dish on the 2nd day except the A30 dish(P0.05). In the osmotic control group (A/B 25) no significant correlation was observed. On the SEM, we could see the separated intercellular junction and fused microvilli of glomerular epithelial cells in the culture dishes where AGE was added. The permeability of BSA increased by 19% only in the A30 dish on the 7th day compared with B5 dish on the 7th day in the permeability assay(P<0.05). CONCLUSION: We observed not only the role of a high level of glucose and AGE in decreasing the production of HSPG of glomerular epithelial cells in vitro, but also their additive effect. However, the role of AGE is greater than that of glucose. These results seems to correlate with the defects in charge selective barrier. Morphological changes of the disruption of intercellular junction and fused microvilli of glomerular epithelial cells seem to correlate with the defects in size-selective barrier. Therefore, we can explain the increased permeability of glomerular epithelial units in the in vitro diabetic condition.

High Glucose and Advanced Glycosylation Endproducts(AGE) Modulate the P-cadherin Expression in Glomerular Epithelial Cells(GEpC)

PURPOSE: Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the integrity of slit diaphragm(SD) proteins including nephrin, P-cadherin, and others. Diabetic proteinuric condition demonstrates defects in SD molecules as well as ultrastructural changes in podocytes. We examined the molecular basis for this alteration of SD molecules especially on P-cadherin as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and AGE induce changes in SD, we cultured rat GEpC under normal(5 mM) or high glucose(30 mM) and AGE- or BSA-added conditions and measured the change of P-cadherin expression by Western blotting and RT- PCR. RESULTS: We found that administration of high glucose decreased the P-cadherin production significantly in the presence or absence of AGE by Western blotting. In RT-PCR high glucose with or without AGE also significantly decreased the expression of P-cadherin mRNA compared to those of controls. Such changes were not seen in the osmotic control. CONCLUSION: We suggest that high glucose with or without AGE suppresses the production of P-cadherin at the transcriptional level and that these changes may explain the functional changes of SD in diabetic conditions.

High Glucose and Advanced Glycosylation Endproducts (AGE) Modulate the CD2AP Expression in Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the foot processes and componental changes in slit diaphragm. We examined the molecular basis for this alteration of the podocyte phenotype, involving quantitative and distributional changes especially on CD2AP as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (HG, 30 mM) and AGE- or BSA-added conditions and examined the distribution of CD2AP by confocal microscope and measured the change of CD2AP expression by Western blotting and RT-PCR. RESULTS: We found that CD2AP moved from peripheral to inner cytoplasm in the HG condition by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the CD2AP productions by 36.9% (p<0.05) and 16.0% (p< 0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of CD2AP more significantly by 64.6% compared to those of control (p<0.01). Such changes was not seen in osmotic control. In RT-PCR, administration of high glucose, AGE or both high glucose and AGE decreased the expression of CD2AP mRNA by 44.9%, 27.9%, and 29.3% (p<0.05), respectively, compared to that of control. CONCLUSION: We could find that HG induce the inward translocation of CD2AP molecule and HG and AGE suppress the production of CD2AP at transcriptional and partly translational level. We suggest that these changes may explain the structural and functional changes of podocytes in diabetic conditions.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on the alpha-actinin-4 Expressed by Glomerular Epithelial Cells (GEpC) / 대한신장학회잡지

BACKGROUND: Regardless of the underlying diagnosis, the proteinuric condition demonstrates ultrastructural changes in GEpC with retraction and effacement of the highly specialized interdigitating podocyte foot processes. I examined the molecular basis for this alteration of the podocyte phenotype, involving cytoskeletal changes especially on alpha-actinin-4 as a candidate regulating the modulation of pathogenic changes in the barrier to protein filtration and regulation of the podocyte actin cytoskeleton. METHODS: To investigate whether high glucose and AGE induce podocyte cytoskeletal changes, we cultured rat GEpC under normal (5 mM) or high glucose (30 mM) and AGE- or BSA-added conditions and examined the distribution of alpha-actinin-4 by confocal microscope and measured the change of alpha-actinin-4 production by Western blotting and RT-PCR. RESULTS: I found that alpha-actinin-4 moved from peripheral cytoplasm to inner actin filaments complexes in the condition of AGE and high glucose by confocal microscopy. In Western blotting, administration of high glucose or AGE decreased the alpha-actinin-4 productions by 22.3% (p>0.05) and 28.1% (p<0.05), respectively. Furthermore, both high glucose and AGE decreased the amount of alpha-actinin-4 more significantly by 53.6% compared to those of control (p<0.01). S uch changes could not be seen by osmotic control. The expression of mRNA for alpha- actinin-4 were not changed in condition of high glucose or AGE-coated surface, however, both high glucose and AGE significantly decreased the expression of alpha-actinin-4 mRNA by 15.7% compared to those of control. CONCLUSION: I could suggest that both high glucose and AGE induce the cytoplasmic translocation and suppress the production of alpha-actinin-4 at transcriptional level and these changes may explain the cytoskeletal changes of GEpC in diabetic conditions.

Effects of High Glucose and Advanced Glycosylation Endproducts (AGE) on ZO-1 Expression in cultured Glomerular Epithelial Cells (GEpC)

PURPOSE: Regardless of the underlying diseases, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. We examined the molecular basis for this alteration of the podocyte phenotypes, including quantitative and distributional changes of ZO-1 protein as a candidate contributing to the pathogenic changes in the barrier to protein filtration. METHODS: To investigate whether high glucose and advanced glycosylation endproduct(AGE) induce podocyte cytoskeletal changes, we cultured rat GEpC under 1) normal glucose(5 mM, =control) or 2) high glucose(30 mM) or 3) AGE-added or 4) high glucose plus AGE-added conditions. The distribution of ZO-1 was observed by confocal microscope and the change of ZO-1 expression was measured by Western blotting and RT-PCR. RESULTS: By confocal microscopy, we observed that ZO-1 moves from peripheral cytoplasm to inner actin filaments complexes in both AGE-added and high glucose condition. In Western blotting, high glucose or AGE-added condition decreased the ZO-1 protein expression by 11.1%(P>0.05) and 2.3%(P>0.05), respectively compared to the normal glucose condition. High glucose plus AGE-added condition further decreased ZO-1 protein expression to statistically significant level(12%, P<0.05). No significant change was seen in the osmotic control. In RT-PCR, high glucose plus AGE-added condition significantly decreased the expression of ZO-1 mRNA by 12% compared to normal glucose condition. CONCLUSION: We suggest that both high glucose and AGE-added condition induce the cytoplasmic translocation and suppresses the production of ZO-1 at transcriptional level and these changes may explain the functional changes of podocytes in diabetic conditions.

Effects of Eligh Glucose and Advance Gilycosylation Endproducts(AGE) on the Heparan Sulfate Proteoglycan(HSPG) Produced by Cultured Rat Clomerular Epithelial Cells(GEC) / 대한신장학회잡지

HSPG, a component of size-and charge-selective barrier of glomerular basement membrane, is one of important matrix proteins which has been known to be reduced in the kidney of diabetic patients or animals. To examine the effects of glucose and AGE on the HSPG production by cultured GEC, we cultured rat GEC on the AGE- or BSA-coated plate under normal(5mM) and high glucose.(30mM) conditions and measured the change of HSPG production by sandwich-ELISA assay and northern blot analysis at 2 days and one week incubation periods. There was no difference in proliferation between 2 different conditions of culture plate surface. We measured the relative amount of the extracted HSPC and observed significant decreases in high glucose condition at one week incubation, and particularly on the AGE-coated surface as compared to the results of BSA-coated condition, by 22% and 5%, respectively. The expression of mRNA for perlecan promoter was decreased in condition of high glucose and AGE-coated surface by 20Yo at 2 days and 61i at one week. Even in normal glucose condition, the expression of mRNA was reduced by 30Yo at one week if the plate was coated with AGE. In conclusion, both high glucose and AGE have reducing effects on the production of HSPG by GEC in vitro. Their effects seem to be additive, however, the role of AGE is greater than that of glucose, This means that the effort to inhibit AGE formation is more important than short-term glucose control for the prevention of diabetic proteinuria.