Pyridoxal phosphate prevents progression of diabetic nephropathy.

BACKGROUND: We have demonstrated that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, inhibits formation of advanced glycation end-products (AGEs) by trapping 3-deoxyglucosone. The present study aimed to clarify if PLP could exert beneficial effects on nephropathy in diabetic rats. METHODS: Streptozotocin (STZ)-induced diabetic rats were treated by oral administration of PLP or pyridoxamine (PM), another active form of vitamin B6, at a dose of 600 mg/kg/day for 16 weeks. AGEs [imidazolone, N(epsilon)-(carboxymethyl)lysine (CML) and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG)], transforming growth factor-beta1 (TGF-beta1), type 1 collagen and fibronectin were detected in the kidneys using immunohistochemistry. Gene expression of TGF-beta1 and receptor for AGEs (RAGEs) in the kidneys was determined using real-time quantitative polymerase chain reaction. RESULTS: Administration of PLP significantly inhibited albuminuria, glomerular hypertrophy, mesangial expansion, and interstitial fibrosis as compared with diabetic rats. PLP markedly inhibited accumulation of AGEs such as imidazolone, CML and CEdG, a DNA-linked AGE, in glomeruli. PLP significantly inhibited expression of TGF-beta1, type 1 collagen, fibronectin and RAGE in the kidneys. PLP was superior to PM in inhibiting accumulation of AGEs, expression of TGF-beta1, type 1 collagen, and fibronectin, and the development of diabetic nephropathy. CONCLUSIONS: PLP prevented progression of nephropathy in STZ-induced diabetic rats by inhibiting formation of AGEs. PLP is considered a promising active form of vitamin B6 for the treatment of AGE-linked disorders such as diabetic nephropathy.     

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)     

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。     

Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.     

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.     

Cultured tubule cells from TGF-beta1 null mice exhibit impaired hypertrophy and fibronectin expression in high glucose

BACKGROUND: To firmly establish the role of the transforming growth factor-beta1 (TGF-beta1) isoform in the pathophysiology of diabetic tubulointerstitial hypertrophy and fibrosis, we examined how the total absence of TGF-beta1 would alter the effect of high glucose on cellular hypertrophy and matrix expression in tubuloepithelial cells cultured from TGF-beta1 null mice. METHODS: Primary tubule cell cultures, obtained from kidneys of TGF-beta1 knockout mice and their wild-type littermates, were treated with exogenous TGF-beta1 or high glucose. The TGF-beta system was characterized at the ligand and receptor levels using Northern and Western blotting. Cellular hypertrophy and growth were assessed by thymidine incorporation, cell counting, leucine incorporation, and protein content. Fibronectin expression was assessed by Northern analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: Knockout cells did not express TGF-beta1 but did express TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors. Exogenous TGF-beta1 down-regulated the ligand-binding type II receptor but up-regulated type I receptor expression. Knockout cells proliferated more rapidly than wild-type cells, but restoring TGF-beta1 to knockout cells slowed their proliferation. In wild-type cells, high glucose caused cellular hypertrophy, evidenced by greater leucine incorporation and protein content along with decreased thymidine incorporation. High glucose also increased fibronectin message and protein. However, in knockout cells, high glucose failed to induce hypertrophy and was severely limited in its capacity to stimulate fibronectin. CONCLUSION: In tubular epithelial cells, TGF-beta1 mediates the hypertrophic and fibronectin-stimulatory effects of high glucose, confirming the role of the TGF-beta1 isoform in the pathogenesis of diabetic tubular hypertrophy and fibronectin overexpression.     

Advanced glycation end products inhibit tubulogenesis and migration of kidney epithelial cells in an ezrin-dependent manner

Nonenzymatic glycation of proteins to form advanced glycation end products (AGE) is implicated in diabetic complications, including nephropathy. It was shown recently that AGE bind to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeletal linker proteins in renal homogenates. Herein is reported the effects of AGE-BSA on ezrin-dependent LLC-PK1 kidney epithelial cellular functions: migration and hepatocyte growth factor (HGF)-induced tubulogenesis. LLC-PK1 cells were stably transfected with cDNA for ezrin sense, ezrin antisense, and N-ezrin. Transfection of LLC-PK1 cells with ezrin antisense and dominant negative N-ezrin decreased basal tubulogenesis and migration relative to vector-only transfection, establishing the ezrin dependency of these processes. AGE-BSA (20 or 40 microM) significantly decreased HGF-induced tubulogenesis and basal migration in two vector control lines relative to BSA-treated cells. However, AGE-BSA inhibition of both HGF-induced tubulogenesis and migration was overcome by overexpressing ezrin. These results demonstrate that the AGE-ezrin interaction significantly alters cellular function. These changes may be relevant to detrimental renal consequences as a result of diabetes.     

晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响

目的：探讨晚期糖化终末产物对人成骨细胞增殖及分泌细胞因子的影响,以期揭示晚期糖化终末产物在骨质疏松发病中的作用。方法：用体外制备的AGE-牛血清白蛋白（AGE-BSA）加入人成骨细胞培养体系,观察不同浓度AGE和不同作用时间,对人成骨细胞增殖及分泌白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的影响。结果：加入AGE-BSA24h后,低浓度抑制成骨细胞增殖;培养48、72h后,浓度依赖性的促增殖作用增加。加入AGE-BSA48h,100μg/ml AGE-BSA促进成骨细胞分泌IL-6。200μg/ml-100μg/ml对IL-6及TNF-α的分泌均无显影响。结论：晚期糖化终末产物对人成骨细胞表现出在较高浓度时,仍具有很强的促增殖活性,提示人对高浓度AGE的耐受性增强。AGE在较高浓度对人成骨细胞分泌IL-6和TNF-α无显影响。提示由随龄增加的AGE在老年人引起的成骨细胞介导的骨吸收没有明显增加,与老年性骨质疏松为低转换型结果相一致。     

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.     

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。     

Immunohistochemical detection of an AGE,a ligand for macrophage receptor,in peritoneum of CAPD patients

BACKGROUND: In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), ultrafiltration (UF) capacity of peritoneal membrane may be impaired due to accumulation of advanced glycation end products (AGEs). This study aimed to elucidate the characteristics of a novel anti-AGE antibody, ODI-GLC19, and to demonstrate AGE accumulation in the peritoneum of CAPD patients using the antibody. METHODS: A monoclonal anti-AGE antibody (ODI-GLC19) was prepared by immunizing female balb/c mice using D-glucose-modified keyhole limpet hemocyanin. The characteristics of ODI-GLC19 were determined by enzyme-linked immunosorbent assay and receptor binding inhibition assay. Immunohistochemistry using ODI-GLC19 was performed to detect AGE in peritoneal tissues obtained from patients with nonrenal disease, and CAPD patients with normal and low UF. RESULTS: ODI-GLC19 reacted with glycolaldehyde-modified BSA (GA-BSA) and glucose-modified BSA (GLC-BSA), but not with imidazolone or N epsilon-(carboxymethyl)lysine. GA-BSA and GLC-BSA strongly bound to cultured macrophages. Time-dependent recognition of newly formed GA-BSA by ODI-GLC19 was similar to that by macrophages. The binding of GA-BSA to macrophages was inhibited by ODI-GLC19 in a dose-dependent manner. Immunohistochemical studies revealed that ODI-GLC19-positive AGE was exclusively detected in peritoneal cells including macrophages, and its staining intensity was more prominent in the peritoneum of CAPD patients, especially with low UF, than in patients with nonrenal disease. CONCLUSIONS: A novel monoclonal anti-AGE antibody, ODI-GLC19, recognizes a ligand for an AGE receptor on macrophages. Incorporation of AGE into peritoneal cells including macrophages may be involved in progressive peritoneal dysfunction in CAPD patients.     

High glucose inhibits renal proximal tubule cell proliferation and involves PKC, oxidative stress, and TGF-beta 1

BACKGROUND: The alteration of renal cell growth is one of the early abnormalities in the diabetic nephropathy. However, the effects of high glucose and its action mechanism in renal proximal tubule cell (PTC) proliferation have not been elucidated. METHODS: The effects of 25 mmol/L glucose on cell proliferation, thymidine, and leucine incorporation, cell cycle, and lipid peroxide formation were examined in the primary cultured renal PTCs. RESULTS: Glucose 25 mmol/L inhibited [3H]-thymidine incorporation and decreased cell growth. However, it increased [3H]-leucine incorporation and protein content. Furthermore, 25 mmol/L glucose increased lipid peroxide formation. These effects of glucose were blocked by antioxidants, vitamin E, N-acetylcystein, or taurine. Staurosporine and H-7 totally blocked 25 mmol/L glucose-induced lipid peroxide formation and had an inhibitory effect on [3H]-thymidine incorporation. Indeed, 25 mmol/L glucose increased the translocation of protein kinase C (PKC) from cytosolic fraction to membrane fraction. In addition, high glucose increased the secretion of transforming growth factor-beta1 (TGF-beta 1) via the PKC-oxidative stress pathway, and TGF-beta 1 inhibited [3H]-thymidine incorporation in a dose-dependent manner. CONCLUSIONS: High glucose inhibits renal PTC proliferation via PKC, oxidative stress, and the TGF-beta 1 signaling pathway.     

Myofibroblast transdifferentiation of mesothelial cells is mediated by RAGE and contributes to peritoneal fibrosis in uraemia.

BACKGROUND: Uraemia is associated with fibrosis of the peritoneal membrane, even prior to the start of peritoneal dialysis. Increased carbonyl stress and the resultant formation of advanced glycation end-products (AGEs) are potentially involved. The interaction of AGEs with their cell surface receptor for AGE (RAGE) induces sustained cellular activation, including the production of the fibrogenic growth factor-beta (TGF-beta). TGF-beta is pivotal in the process of epithelial-to-mesenchymal transition with the acquisition of myofibroblast characteristics. We investigated whether antagonism of RAGE prevents uraemia-induced peritoneal fibrosis. In addition, we examined whether myofibroblast transdifferentiation of mesothelial cells contributes to peritoneal fibrosis in uraemia. METHODS: Uraemia was induced in rats by subtotal nephrectomy. Uraemic and age-matched sham-operated rats were treated for 6 weeks with neutralizing monoclonal anti-RAGE antibodies or placebo. Expression of AGE, RAGE, cytokeratin and alpha-smooth muscle actin was evaluated using immunohistochemistry. TGF-beta expression was examined with immunostaining and western blotting, and Snail expression with western blotting. Fibrosis was quantified with a picro-sirius red staining and measurement of the hydroxyproline content of the tissue. RESULTS: Uraemia resulted in the accumulation of AGE, up-regulation of RAGE and TGF-beta and the development of interstitial fibrosis and vascular sclerosis in the peritoneal membrane. Prominent myofibroblast transdifferentiation of mesothelial cells was identified by colocalization of cytokeratin and alpha-smooth muscle actin in submesothelial and interstitial fibrotic tissue. The antagonism of RAGE prevented the up-regulation of TGF-beta, epithelial-to-mesenchymal transition of mesothelial cells and fibrosis in uraemia. CONCLUSION: The ligand engagement of RAGE and the subsequent up-regulation of TGF-beta induces peritoneal fibrosis in chronic uraemia. The process may be mediated by the conversion of mesothelial cells into myofibroblasts.     

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb uptake in pig renal proximal tubule cell line (LLC-PK1) cells; and ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells in a clathrin-dependent pathway. Our data also suggest a role of endocytosis in both ouabain-induced signal transduction and proximal tubule sodium handling. The present study addresses the molecular mechanisms involved in this process. METHODS: Studies were performed with cultured LLC-PK1 and a stable-expressed caveolin-1 knockdown LLC-PK1 cell line by SiRNA method. RESULTS: In wild-type LLC-PK1 cells, depletion of cholesterol by methyl beta-cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coated vesicles, as well as in endosomes. Depletion of cholesterol also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. In LLC-PK1 cells expressing mock-vehicle and caveolin-1 siRNA, depletion of caveolin-1 abolished ouabain-induced decrease in Rb uptake and decrease in the plasmalemmal Na/K-ATPase content. Depletion of caveolin-1 also significantly reduced the ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coat vesicles, as well as early and late endosomes. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. These data suggest that caveolae are involved in ouabain-induced endocytosis and signal transduction by initiating assembly of signaling cascades through the caveolar Na/K-ATPase and/or the interaction with clathrin-mediated endocytosis of the Na/K-ATPase.     

Effects of advanced glycation end products on cytosolic Ca2+ signaling of cultured human mesangial cells.

Advanced glycation end product (AGE) accumulation in a high glucose (HG) environment is thought to mediate some of the vascular complications of diabetes. Transmembrane signaling of contractile cells is generally inhibited by HG, with implications for systemic and target organ hemodynamics. In the kidney, glomerular mesangial cells grown in HG media are hyporesponsive to the effects of vasoconstrictor agents, possibly explaining the hyperfiltration and increased capillary pressure that eventually lead to diabetic glomerulopathy. To verify whether AGE binding to specific mesangial receptors could mediate these effects of HG, cultured human mesangial cells (HMC) were exposed to in vitro glycated bovine serum albumin (BSA) for 60 min at 37 degrees C before measurement of cytosolic Ca2+ ([Ca2+]i) by microfluorometric techniques in monolayers or single cells. AGE-BSA (2 mg/ml) reduced Ca2+ release from intracellular stores by 1 microM angiotensin II from peak [Ca2+]i levels of 843+/-117 to 390+/-50 nM in monolayers and from 689+/-68 to 291+/-36 nM in individual cells (P < 0.05). Nonglycated BSA and BSA exposed to 250 mM glucose-6-phosphate for 30 d in the presence of 250 mM aminoguanidine (AMGD), an inhibitor of nonenzymatic glycation, had no effect on the angiotensin II-induced [Ca2+]i spike (peak 766+/-104 and 647+/-87 nM, monolayers/ single cells, respectively, P = NS). AGE also inhibited store-operated Ca2+ influx through plasma membrane channels, assessed by addition of 1 to 10 mM extracellular Ca2+ to cells previously held in Ca2(+)-free media (control 339+/- 46/593 +/- 51, +AGE-BSA 236 +/- 25/390 +/- 56, +AMGD 483+/-55/ 374+/-64 nM [Ca2+]i, monolayers/single cells at 10 mM Ca2+, respectively; +AGE-BSA, P < 0.05 versus control). Contrary to HG, AGE-BSA did not translocate protein kinase C isoforms alpha, zeta, and delta to the plasma membrane. Culture of HMC in HG supplemented with 1 mM AMGD prevented downregulation of [Ca2+]i signaling. These data suggest that glycated macromolecules or matrix components may inhibit transmembrane Ca2+ signaling of glomerular cells through binding to a specific AGE receptor, thus mediating some of the known functional effects of HG on the kidney.     

晚期糖基化终产物诱导人肾小管上皮细胞表达结缔组织生长因子

目的观察晚期糖基化终产物（AGE）修饰的牛血清白蛋白（BSA）对体外培养的人近端肾小管上皮细胞株（HK-2细胞）表达结缔组织生长因子（CTGF）的影响，并探讨可能的作用途径。方法将HK-2细胞在体外与不同浓度的AGE-BSA和BSA共同培养。间接免疫荧光法检测糖基化终产物受体（RAGE）。免疫印迹法观察HK-2细胞表达的CTGF蛋白质，ELISA和免疫印迹法检测TGF-β1的合成和分泌。结果在HK-2细胞的胞膜和胞质存在RAGE的表达。AGE-BSA以时间和剂量依赖方式上调HK-2细胞CTGF的表达，8、24、48及72h组分别为0h组的117％、138％、257％及339％：20、50及100mg／L组分别为0mg／L组的186％、240％及287％。在一定的剂量和时间范围内，AGE—BSA能够诱导TGF-β1蛋白质的表达和分泌。抗RAGE和抗TGF-β1的单克隆抗体能够部分抑制AGE-BSA诱导的CTGF蛋白质表达。结论人肾小管上皮细胞表面存在RAGE的表达。AGE—BSA可能部分地通过RAGE介导TGF-β1表达上调。诱导HK-2细胞CTGF表达。     

Cell cycle inhibitors (p27Kip1 and p21CIP1) cause hypertrophy in LLC-PK1 cells.

BACKGROUND: Angiotensin II has been reported to induce renal tubular hypertrophy, but the mechanisms of this hypertrophy are not well known. We evaluated the roles of cyclin-dependent kinase (CDK) inhibitors in renal tubular hypertrophy. METHODS: To elucidate whether CDK inhibitors cause renal tubular hypertrophy, we produced adenovirus vectors containing coding sequences of the CDK inhibitors p27Kip1 (AxCAp27), p21CIP1 (AxCAp21), and p16INK4 (AxCAp16), and we investigated the effect of these gene transfers on epidermal growth factor (EGF)-induced proliferation in LLC-PK1 cells. We evaluated the cell cycle and hypertrophy by measurements of the [3H]-leucine and [3H]-thymidine incorporation, the protein:DNA ratio, flow cytometry, and CDK4 and CDK2 kinase assays. RESULTS: AxCAp27 and AxCAp21 caused significant increases in [3H]-leucine incorporation and the protein:DNA ratio but did not change the [3H]-thymidine incorporation. Conversely, AxCAp16 inhibited EGF-stimulated [3H]-thymidine incorporation but did not change the [3H]-leucine incorporation. AxCAp27, AxCAp21, and AxCAp16 all inhibited EGF-stimulated CDK4 kinase activity (to 15.6, 14.1, and 21.9% of control, respectively). Forward light-scatter analysis demonstrated that AxCAp27 and AxCAp21 increased the cell size but that AxCAp16 effected no change in cell size. CONCLUSION: These findings suggest that p27Kip1 and p21CIP1 may play an important role in hypertrophy of renal tubule cells by reducing pRb phosphorylation. On the other hand, p16INK4 was not found to cause hypertrophic changes in EGF-treated LLC-PK1 cells.