A Receptor‐Based Bioadsorbent to Target Advanced Glycation End Products in Chronic Kidney Disease

The accumulation of advanced glycation end products (AGEs) has been reported to be a major contributor to chronic systemic inflammation. AGEs are not efficiently removed by hemodialysis or the kidney of a chronic kidney disease (CKD) patient. The goal of this study was to develop a receptor for AGEs (RAGE)‐based bioadsorbent device that was capable of removing endogenous AGEs from human blood. The extracellular domain of RAGE was immobilized onto agarose beads to generate the bioadsorbent. The efficacy of AGE removal from saline, serum, and whole blood; biological effects of AGE reduction; and hemocompatibility and stability of the bioadsorbent were investigated. The bioadsorbent bound AGE‐modified bovine serum albumin (AGE‐BSA) with a binding capacity of 0.73 ± 0.07 mg AGE‐BSA/mL bioadsorbent. The bioadsorbent significantly reduced the concentration of total AGEs in serum isolated from end‐stage kidney disease patients by 57%. AGE removal resulted in a significant reduction of vascular cell adhesion molecule‐1 expression in human endothelial cells and abolishment of osteoclast formation in osteoclast progenitor cells. A hollow fiber device loaded with bioadsorbent‐reduced endogenous AGEs from recirculated blood to 36% of baseline levels with no significant changes in total protein or albumin concentration. The bioadsorbent maintained AGE‐specific binding capacity after freeze‐drying and storage for 1 year. This approach provides the foundation for further development of soluble RAGE‐based extracorporeal therapies to selectively deplete serum AGEs from human blood and decrease inflammation in patients with diabetes and/or CKD.     

Differential effects of advanced glycation end-products on renal tubular cell inflammation

Aim: The authors recently showed that advanced glycation end‐products (AGE) in the form of glycated albumin (GA) upregulated renal tubular expression of interleukin (IL)‐8 and soluble intercellular adhesion molecule‐1 (sICAM‐1), but not other important cytokines known to mediate diabetic nephropathy. This implies that other molecules such as the carbonyl intermediates of AGE or other modified protein lysine‐albumin may participate in diabetic tubular injury. Methods: Human proximal tubular epithelial cells (PTEC) were growth‐arrested and exposed to methylglyoxal (MG), MG‐bovine serum albumin (BSA)‐AGE, carboxymethyllysine (CML)‐BSA, AGE‐BSA or BSA with or without prior addition of rosiglitazone that was previously shown to attenuate the pro‐inflammatory effect of GA alone. Results: MG‐BSA‐AGE and AGE‐BSA upregulated tubular expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)‐β, and vascular endothelial growth factor (VEGF), whereas CML‐BSA stimulated expression of IL‐6, CCL‐2, CTGF, TGF‐β and VEGF. These AGE compounds also activated nuclear factor (NF)‐κB and their effects were attenuated by pre‐incubation with anti‐RAGE antibody. MG and BSA did not affect the expression of any of these molecules. Rosiglitazone did not affect the in vitro biological effects of MG, MG‐BSA‐AGE, AGE‐BSA or CML‐BSA on PTEC. Conclusion: AGE exhibit differential inflammatory and fibrotic effects on PTEC via RAGE activation and NF‐κB signal transduction. Rosiglitazone had no effect on these responses. Further investigations on compounds that nullify the downstream effects of these AGE are warranted.     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin

Background: Cell-mediated immunity is impaired in uraemia. The recognition and ensuing interactions of immune cells, such as CD4+ T lymphocytes, with adhesive glycoproteins of the extra-cellular matrix (ECM) are mediated by integrins of the {beta}1 subfamily. We have previously demonstrated that uraemic sera inhibit the proliferation and adhesion of normal CD+ T cells to ECM components. In the present study, the adhesive capacity of CD4+ T lymphocytes of dialyzed patients (both haemodialysis [HD] and continuous ambulatory peritoneal dialysis [CAPD] was evaluated. Methods: Adhesion of CD4+ T cells from dialysis patients to intact ECM and its immobilized moieties, fibronectin (FN) and laminin (LN) was measured following phorbol-12-myristate-13-acetate (PMA) stimulation. In addition, cell surface expression of {beta}1 integrins (VLA 4-6) was determined by FACScan analysis. Results: Compared to normal cells, CD4+ T cells of dialysis patients demonstrated a significantly reduced adhesion to ECM, FN and LN (27-28 vs 52-55% P <0.001). This decreased adhesive capacity was not normalized upon incubation of the cells with normal sera. Cell surface expression of {beta}1 integrins was not modified. The inhibition of cell adhesion was more pronounced in CAPD patients (23-24% vs 29-30% in HD, P <0.02). Serum albumin correlated directly with cell adhesion. Aged HD patients' T cells demonstrated increased adhesion to ECM and its ligands, whereas a reverse trend was demonstrated in the CAPD group. Conclusions: T cells of dialysis patients exhibit abnormal adhesive activity, which may be due to an acquired cellular defect induced by uraemic milieu. CAPD patients show a greater degree of adhesion impairment, possibly due to their lower concentrations of serum albumin.     

Radioreceptor assay for advanced glycosylation end products.

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1 mu CI/ml [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 micrograms/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid-BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

终末期糖基化终产物通过转化生长因子依赖和非依赖途径介导NRK52E细胞转分化和胶原Ⅰ的合成

目的探讨终末期糖基化终产物（AGEs）介导肾小管上皮细胞转分化和胶原（Col）Ⅰ合成的分子机制。方法体外培养正常大鼠近端肾小管上皮细胞系（NRK52E），应用自制的AGE-牛血清白蛋白（BSA）刺激NRK52E细胞。免疫细胞化学方法检测不同时间磷酸化（P）Smad2／3核转位情况。ELISA方法检测细胞培养上清TGF-β1的水平。RT-PCR方法检测α-平滑肌肌动蛋白（SMA）、E-钙黏着糖蛋白（cadherin）和ColⅠmRNA表达。Western印迹检测α-SMA、E-cadherin和ColⅠ蛋白的表达。同时观察TGF-β1中和抗体对AGE-BSA上述效应的阻断作用。结果基础状态下，NRK52E细胞存在低水平p-Smad2／3核表达（16％）。与BSA对照组比较，AGE—BSA以时间依赖方式上调NRK52E细胞p-Smad2／3核转位，其高峰出现在30min（68％比30.5％，P〈0．01）和24h（76％比31．3％，P〈0．01）。AGE—BSA显著上调α-SMA和ColⅠmRNA和蛋白表达；下调E-cadherin mRNA和蛋白的表达；并能促进NRK52E细胞合成和分泌TGF-β1。TGF-β1中和抗体能明显抑制AGE—BSA介导的24hp-Smad2／3核转位（25．2％，P〈0．01），但不能阻抑30min活化高峰；能明显抑制AGE-BSA介导的α-SMA和ColⅠmRNA和蛋白表达．以及显著地上调E-cadherin mRNA和蛋白的表达。结论AGEs通过TGF-β依赖和非依赖途径诱导肾小管上皮细胞Smads信号通路活化，促进其向肌成纤维母细胞转分化和ColⅠ的合成。     

Advanced glycation end-products attenuate human mesenchymal stem cells and prevent cognate differentiation into adipose tissue, cartilage, and bone.

The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients. INTRODUCTION: Advanced glycation end-products (AGEs) are accumulated on long-lived proteins of various tissues in advanced age and diabetes mellitus and have been implicated in chronic complication, including musculoskeletal disorders. Human mesenchymal stem cells (MSCs) potentially differentiate into mature musculoskeletal tissues during tissue repair, but the pathogenetic role of AGEs on MSCs is unclear. MATERIALS AND METHODS: AGEs were prepared by incubating BSA with glucose, glyceraldehydes, or glycolaldehyde (designated as AGE-1, AGE-2, or AGE-3, respectively). Proliferation, apoptosis, and reactive oxygen species (ROS) generation were assayed in AGE-treated cells. The expression of the receptor for AGE (RAGE) was examined by immunohistochemistry and Western blotting. Involvement of RAGE-mediated signaling was examined using a neutralizing antiserum against RAGE. Differentiation into adipose tissue, cartilage, and bone were morphologically and biochemically monitored with specific markers for each. RESULTS: AGE-2 and AGE-3, but not control nonglycated BSA and AGE-1, reduced the viable cell number and 5-bromo-2'deoxyuridine (BrdU) incorporation with increased intracellular ROS generation and the percentage of apoptotic cells. MSCs expressed RAGE and its induction was stimulated by AGE-2 and AGE-3. These AGEs inhibited adipogenic differentiation (assayed by oil red O staining, lipoprotein lipase production, and intracellular triglyceride content) and chondrogenic differentiation (assayed by safranin O staining and type II collagen production). On osteogenic differentiation, AGE-2 and AGE-3 increased alkaline phosphatase activity and intracellular calcium content; however, von Kossa staining revealed the loss of mineralization and mature bone nodule formation. The antiserum against RAGE partially prevented AGE-induced cellular events. CONCLUSION: AGE-2 and AGE-3 may lead to the in vivo loss of MSC mass and the delay of tissue repair by inhibiting the maturation of MSC-derived cells. The AGE-RAGE interaction may be involved in the deleterious effect of AGEs on MSCs.     

Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs) that are recognized by AGE receptors. Glyoxal, glycolaldehyde (GA), and methylglyoxal are potential intermediates for the formation of AGE structures such as Nomega-(carboxymethyl)lysine (CML). We evaluated the contribution of these aldehydes to the formation of AGE structure(s), particularly the structure important for the receptor-mediated endocytic uptake of AGE proteins by macrophages. GA-modified bovine serum albumin (BSA), methylglyoxal-modified BSA (MG-BSA), and glyoxal-modified BSA (GO-BSA) were prepared, and their physicochemical, immunological, and biologic properties were compared with those of glucose-derived AGE-BSA. CML contents were high in GO-BSA and low in GA-modified BSA (GA-BSA) but did not exist in MG-BSA. The fluorescence patterns of GA-BSA and MG-BSA were similar to those of glucose-derived AGE-BSA but were weak in GO-BSA. Immunochemically, the antibody against non-CML structures of glucose-derived AGE-BSA reacted strongly with GA-BSA and weakly with GO-BSA but did not react with MG-BSA. The negative charge of these ligands increased to a similar extent. However, GA-BSA, but not MG-BSA or GO-BSA, underwent receptor-mediated endocytosis by the macrophage-derived cell line RAW 264.7, which was effectively inhibited by glucose-derived AGE-BSA, acetylated LDL, and oxidized LDL, which are well-known ligands for the macrophage type I and type II class A scavenger receptors (MSR-A). The endocytic uptake of GA-BSA by mouse peritoneal macrophages was also significant, but that by peritoneal macrophages from MSR-A-deficient mice was markedly reduced. Our results suggest that GA serves as an important intermediate for the generation of AGE structure(s) responsible for recognition by MSR-A.     

Glycation, glycoxidation and diabetes mellitus

Advanced glycation end-products (AGEs) result from a reaction between carbohydrates and the free amino groups of proteins, lipids, and DNA. Non enzymatic glycation, glycoxidation with glucose auto-oxidation and the polyol pathway are involved in glycated protein formation. AGEs also named glycotoxins are found in excess in pathological situations such as diabetes mellitus, renal failure, and aging or after absorption of food containing glycated products. Three major pathophysiological mechanisms are described to explain AGE toxicity, first AGEs can accumulate in the vessel wall and in collagen of different tissues; second in situ glycation is possible; third, AGEs bind to cell receptors inducing deleterious consequences. AGE receptor RAGE is a multiligand member of the immunoglobulin superfamily of cell surface molecules. AGE-receptor interaction can alter, macrophage, endothelial cell, mesangial and mesothelial cell functions and can induce inflammation. Oxidant stress, vascular hyperpermeability, vascular cell adhesion molecule-1 (VCAM-1) overexpression and monocytes chemotactic Protein-1 (MCP-1) production have been observed after cell activation by AGEs. AGEs appear to be involved in the genesis of diabetic macro but also microangiopathy such as retinopathy and glomerulosclerosis. New drugs are tested to prevent or break the AGE-protein cross-linkage, or to control the AGE-receptor interaction and their consequences. Dietary treatment, strict glycemic control and preservation of renal function remain the best approach for preventing AGE formation and limiting their deleterious effects.     

Alendronate Reduces Adhesion of Human Osteoclast-like Cells to Bone and Bone Protein-Coated Surfaces

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs. controls. The effect of ALN on the adhesion of osteoclast-like cells onto specific extracellular matrix proteins, such as bone sialoprotein-derived peptide, containing the RGD sequence, conjugated to BSA (BSP-BSA) and fibronectin (FN), was also tested. In the case of FN the treatment with ALN of protein-coated wells did not modify the percentage of cell adhesion compared with the control, whereas onto BSP-BSA the presence of ALN significantly reduced adhesion of about 40–45%, suggesting that the inhibitory effect of ALN on cell adhesion could probably be due to the interference with receptors specifically recognizing bone matrix proteins as α_Vβ₃ integrins. Furthermore, ALN induced Ca-mediated intracellular signals in osteoclasts, triggering a 2-fold increase in intracellular calcium concentration. Received: 8 August 1997 / Accepted: 27 January 1998     

The AGE inhibitor pyridoxamine inhibits lipemia and development of renal and vascular disease in Zucker obese rats

BACKGROUND: In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. METHODS: Three groups of Zucker rats were studied: lean (Fa/fa), untreated fatty (fa/fa), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. RESULTS: AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. CONCLUSION: Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.     

GDF‐5/7 and bFGF activate integrin α2‐mediated cellular migration in rabbit ligament fibroblasts

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein‐2, growth and differentiation factor (GDF)‐5, and GDF‐7 treatment was similar to that of MCL cells. GDF‐5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7‐ and 17‐fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF‐5 than ACL cells. GDF‐5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin α2. Functional blocking analyses using anti‐integrin α2 antibodies revealed that cellular migration responding to growth factors depended on the integrin α2‐mediated adhesion on type I collagen. The expression of integrin α2 was also increased by growth factors in both cells. Our results demonstrate that GDF‐5/7 and bFGF stimulate cellular migration by modulating integrin α2 expression and integrin α2‐dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin α2‐mediated cellular migration in response to growth factors. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:225–231, 2010     

Inhibition by uraemic sera of CD4+ T-cell adhesion to extracellular matrix components

BACKGROUND.: T-cell-mediated immune responses are impaired in patients withchronic renal failure. The migration, proliferation, differentiation,biological functioning, and interaction with other T cells aremediated by cell surface adhesion proteins, which include integrins. METHODS.: To elucidate how uraemia can impair T-cell-mediated responsesin vivo, the effects of sera from uraemic patients on T-cellproliferation and adhesion to extracellular matrix (ECM) componentswere examined. RESULTS.: Preincubation of human CD4⁺ T cells with sera from undialysedand dialysed (haemodialysis or peritoneal dialysis) uraemicpatients inhibited the capacity of the cells to be stimulatedby phytohaemmagglutinin and by anti-CD3 monoclonal antibodyplus immobilized fibronectin (FN). Sera from uraemic and dialysedpatients, but not from healthy individuals, inhibited significantly,and in a dose-dependent fashion, human CD4⁺ T cell adhesionto immobilized FN and laminin (LN). The degree of inhibitionof adhesion was similar whether the sera were continuously present,even during the adhesion assay, or removed by washing. The adhesioninhibiting capacity of the uraemic sera was not due to modificationof the expression of ß1 integrins on the surfacesof the T cells. CONCLUSIONS.: These results suggest that uraemia can impair the proliferativecapacity and adhesion of immune cells, and thus may affect normalimmune processes and contribute to the overall immune deficiencyobserved in patients with renal failure.     

In Vitro-Induced High Sugar Environments Deteriorate Human Cortical Bone Elastic Modulus and Fracture Toughness

Advanced glycation end-products (AGEs) have been suggested to contribute to bone fragility in type 2 diabetes (T2D). AGEs can be induced through in vitro sugar incubations but there is limited data on the effect of total fluorescent AGEs on mechanical properties of human cortical bone, which may have altered characteristics in T2D. Thus, to examine the effect of AGEs on bone directly in T2D patients with uncontrolled sugar levels, it is essential to first understand the fundamental mechanisms by studying the effects of controlled in vitro-induced AGEs on cortical bone mechanical behavior. Here, human cortical bone specimens from female cadaveric tibias (ages 57–87) were incubated in an in vitro 0.6 M ribose or vehicle solution (n = 20/group) for 10 days at 37°C, their mechanical properties were assessed by microindentation and fracture toughness tests, and induced AGE levels were quantified through a fluorometric assay. Results indicated that ribose-incubated bone had significantly more AGEs (+81%, p ≤ 0.005), lower elastic modulus assessed by traditional microindentation, and lower fracture toughness compared with vehicle controls. Furthermore, based on pooled data, increased AGEs were significantly correlated with deteriorated mechanical properties. The findings presented here show that the accumulation of AGEs allows for lower stiffness and increased ability to initiate a crack in human cortical bone. Statement of clinical significance: High sugar levels as in T2D results in deteriorated bone quality via AGE accumulation with a consequent weakening in bone's mechanical integrity. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:972-983, 2020     

Effects of physiologic albumin and hespan levels on hepatocytes in vitro

BACKGROUND: Although albumin and hydroxyethyl starch (HES) are routinely used in critically ill, hypoalbuminemic patients, no studies have tested the effect of supplemental albumin and HES on hepatocyte function. METHODS: In this study, the effects of these agents were evaluated by using stable, rat hepatocyte cultures in a collagen sandwich configuration. Hepatocyte synthesis of albumin, urea, and intracellular triglycerides was monitored in Dulbecco's modified Eagle medium (supplemented with fetal bovine serum, hydrocortisone, L-proline, gentamycin, and insulin) without supplemental colloid (control cultures) and with supplemental 2% bovine serum albumin (BSA), 4% BSA, 2% HES, or 4% HES. RESULTS: The albumin secretion in control cultures rose from 31.03 microg/day per 10(6) cells on day 3 to 154.17 microg/day per 10(6) cells by day 12 and remained constant. In contrast, the level of albumin synthesis in the 2% and 4% BSA groups rose from significantly higher initial values (p < 0.05) of 71.25 microg/day per 10(6) cells and 73.27 microg/day per 10(6) cells, respectively, to 127.61 microg/day per 10(6) cells and 107.95 microg/day per 10(6) cells by day 7, then declined rapidly to 58.98 microg/day per 10(6) cells and 41.28 microg/day per 10(6) cells by day 12 when cell disruption was present. HES also reduced albumin synthesis. The urea genesis in the control groups and in the treatment groups was found to be comparable throughout the study. The BSA supplemented groups accumulated large amounts of intracellular lipid droplets during the experiment. The intracellular triglycerides analysis found the 4% BSA group to be significantly (p < 0.05) higher than the 4% HES. CONCLUSION: BSA, added to a collagen sandwich hepatocyte preparation, causes reduced hepatocyte synthesis by day 8, probably a result of intracellular triglyceride accumulation, whereas HES reduces synthesis through unidentified mechanisms.     

Advanced glycation end product levels in eye lenses, aorta, and tail tendon in transplanted diabetic inbred Lewis rats.

BACKGROUND: Pancreatic islet transplantation in diabetes, by restoring euglycemia, should in time correct the abnormal accumulation of advanced glycation end products (AGEs) over target tissues, thus delaying the development of late diabetic complications. METHODS: Homologous islet transplantation was performed in inbred Lewis rats 15 days (TA), 4 months (TB), and 8 months (TC) after streptozotocin diabetes. Group TA was studied for 12 months and groups TB and TC were studied for 4 months after transplantation. Normal (N) and diabetic (D) rats formed the control groups. Metabolic control in the transplant (T) groups was evaluated by oral glucose tolerance test. Blood glucose, glycated hemoglobin, and body weight were determined in all groups. AGE levels were determined by spectrofluorometry in eye lens proteins and by ELISA in aortic and tail tendon collagen. RESULTS: T groups showed normal oral glucose tolerance tests and metabolic parameters. The latter were altered in all D groups (P<0.005 to P<0.0001 versus N and T groups). AGEs were increased in the D groups (P<0.05 to P<0.001) versus the N groups. AGEs in the TA and TB groups were not different from those of the N groups but were significantly reduced (P<0.05 to P<0.001) when compared with those of the D groups. In the TC group, eye lens AGEs were significantly elevated (P<0.001) or significantly reduced (P<0.01) when compared with those of the N or D groups, respectively. Aortic collagen AGEs were elevated (P<0.01) by comparison with those of the N groups and not statistically different from those of the D groups. Tail tendon collagen AGE levels lay between those of the N and D groups, without reaching a statistical significance. CONCLUSIONS: These results indicate that primary and early secondary (groups TA and TB) but not late secondary (group TC) islet transplantations are capable of blocking or reducing an abnormal accumulation of AGEs, thus confirming the importance of preventive transplantation therapies.