High glucose stimulates angiotensinogen gene expression via reactive oxygen species generation in rat kidney proximal tubular cells

The present studies investigated whether the effect of high glucose levels on angiotensinogen (ANG) gene expression in kidney proximal tubular cells is mediated via reactive oxygen species (ROS) generation and p38 MAPK activation. Rat immortalized renal proximal tubular cells (IRPTCs) were cultured in monolayer. Cellular ROS generation and p38 MAPK phosphorylation were assessed by lucigenin assay and Western blot analysis, respectively. The levels of immunoreactive rat ANG secreted into the media and cellular ANG mRNA were determined by a specific RIA and RT-PCR, respectively. High glucose (25 mM) evoked ROS generation and p38 MAPK phosphorylation as well as stimulated immunoreactive rat ANG secretion and ANG mRNA expression in IRPTCs. These effects of high glucose were blocked by antioxidants (taurine and tiron), inhibitors of mitochondrial electron transport chain complex I (rotenone) and II (thenoyltrifluoroacetone), an inhibitor of glycolysis-derived pyruvate transport into mitochondria (alpha-cyano-4-hydroxycinnamic acid), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a manganese superoxide dismutase mimetic, catalase, and a specific inhibitor of p38 MAPK (SB 203580), but were not affected by an inhibitor of the malate-aspartate shuttle (aminooxyacetate acid). Hydrogen peroxide (>/=10(-5) M) also stimulated p38 MAPK phosphorylation, ANG secretion, and ANG mRNA gene expression, but its stimulatory effect was blocked by catalase and SB 203580. These studies demonstrate that the stimulatory action of high glucose on ANG gene expression in IRPTCs is mediated at least in part via ROS generation and subsequent p38 MAPK activation.     

High levels of glucose stimulate angiotensinogen gene expression via the P38 mitogen-activated protein kinase pathway in rat kidney proximal tubular cells

The present studies investigated whether the effect of high levels of glucose on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells is mediated at least in part via the activation of p38 mitogen-activated protein kinase (p38 MAPK). Rat immortalized renal proximal tubular cells (IRPTCs) were cultured in monolayer. The levels of immunoreactive rat ANG (IR-rANG) secreted into the medium and the levels of cellular ANG messenger RNA were determined by a specific RIA for rat ANG and a RT-PCR assay, respectively. Phosphorylation of cellular p38 MAPK was determined by Western blot analysis using the Phospho Plus p38 MAPK antibody kit. High levels of glucose (i.e. 25 mM) and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) increased the secretion of IR-rANG and cellular ANG messenger RNA as well as phosphorylation of p38 MAPK in IRPTCs. This stimulatory effect of high levels of glucose and PMA was blocked by SB 203580 (a specific inhibitor of p38 MAPK), but not by SB 202474 (a negative control of SB 203580). High levels of D-sorbitol or 2-deoxy-D-glucose (i.e. > or = 35 mM) also stimulated the phosphorylation of p38 MAPK, but did not stimulate ANG secretion or gene expression. GF 109203X (an inhibitor of protein kinase C) blocked the stimulatory effect of high levels of glucose and PMA on ANG gene expression, whereas it did not block the effect of high levels of glucose, sorbitol, or 2-deoxy-D-glucose on p38 MAPK phosphorylation in IRPTCs. These studies demonstrate that the stimulatory effect of a high level of glucose (25 mM) on ANG gene expression in IRPTCS may be mediated at least in part via activation of p38 MAPK signal transduction pathway and is protein kinase C independent.     

Insulin inhibits angiotensinogen gene expression via the mitogen-activated protein kinase pathway in rat kidney proximal tubular cells.

The present study aimed to investigate the molecular mechanism(s) of insulin action on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells exposed to high levels of glucose. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of rat ANG and ANG messenger RNA in the IRPTC were quantified by a specific RIA for rat ANG (RIA-rANG) and by an RT-PCR assay. Insulin inhibited the stimulatory effect of a high level of glucose (25 mM) and phorbol 12-myristate 13-acetate, an activator of protein kinase C) on the secretion of ANG and the expression of the ANG messenger RNA in IRPTC. This inhibitory action of insulin on the ANG secretion and gene expression was blocked by PD98059 (an inhibitor of mitogen-activated protein kinase kinase) but not by Wortmannin (an inhibitor of phosphatidylinositol-3-kinase). PD98059 was effective in inhibiting the phosphorylation of MEK 1/2 and p44/42 MAP kinase in IRPTC stimulated by insulin. These studies demonstrate that insulin prevents the stimulatory effect of high levels of glucose on the expression of the renal ANG gene in IRPTC, at least in part, via the MAPK kinase signal transduction pathway, subsequently inhibiting the activation of the local renal renin-angiotensin system.     

Insulin inhibits dexamethasone effect on angiotensinogen gene expression and induction of hypertrophy in rat kidney proximal tubular cells in high glucose

The present studies investigated whether insulin inhibits the stimulatory effect of dexamethasone (DEX) on angiotensinogen (ANG) gene expression and induction of hypertrophy in rat immortalized renal proximal tubular cells (IRPTCs) in a high-glucose milieu. Rat IRPTCs were cultured in monolayer. ANG and ANG mRNA expression in IRPTCs were quantified by a specific RIA for rat ANG and by RT-PCR assay, respectively. A fusion gene containing the full length of the 5'-flanking region of the rat ANG gene linked to a chloramphenicol acetyl transferase reporter gene was introduced into IRPTCs. The level of fusion gene expression was determined by cellular chloramphenicol acetyl transferase enzymatic activity. Cellular hypertrophy was assessed by flow cytometry, cellular p27(Kip1) protein expression, and protein assay. Our results showed that high glucose (i.e. 25 mM) and DEX (10(-7) M) additively stimulated ANG gene expression and induced IRPTC hypertrophy. Insulin inhibited the effect of high glucose and DEX on these parameters. The inhibitory effect of insulin was reversed by PD 98059 (a MAPK inhibitor) but not by wortmannin (a phosphatidylinositol-3-kinase inhibitor). These results demonstrate that insulin is effective in blocking the stimulatory action of high glucose and DEX on ANG gene expression and induction of IRPTC hypertrophy, suggesting its important role in preventing local intrarenal renin-angiotensin system activation and renal proximal tubular cell hypertrophy induced by hyperglycemia and glucocorticoids in vivo.     

葡萄糖转运蛋白对大鼠肾小球系膜细胞己糖胺通路的影响

目的探讨己糖胺通路(HBP)在葡萄糖转运蛋白1(GLUT1)基因转染系膜细胞功能改变中的作用.方法利用逆转录病毒载体建立GLUT1基因转染的大鼠系膜细胞,以β-半乳糖苷酶转染细胞(MCLacZ)为对照.用2-脱氧-3H-葡萄糖(2-DG)测定细胞葡萄糖摄入,流式细胞仪分析细胞表型和纤维连接蛋白(FN)的合成,采用比色法测定HBP限速酶--谷氨酰胺6-磷酸果糖转氨酶(GFAT)的活性,RT-PCR检测细胞GFAT基因的表达.结果 MCGT1的2-DG摄入率明显高于MCLacZ[(741.0±60.5)dpm/μg蛋白质 vs (92.2±9.0)dpm/μg 蛋白质,P＜0.01],动力学分析发现MCGT1的Vmax是MCLacZ的3.7倍,而两者Km值无明显差别.MCGT1的GFAT活性明显高于MCLacZ[(3.25±0.25)OD365·μg蛋白质-1·30 min-1·10 vs (1.15±0.16)OD365·μg蛋白质-1·30 min-1·     

JAK/STAT信号途径在高糖诱导肾小管上皮细胞转分化中的作用

目的观察JAK/STAT信号途径对高糖诱导肾小管上皮细胞转分化的影响。方法体外培养人肾近曲小管上皮细胞株(HKC),分别给予高糖和JAK抑制剂AG490干预,Western印迹检测α-SMA、E-Cadherin及STAT1、STAT3、p-STAT1和p-STAT3的表达;ELISA法测定上清液中TGF-β1、I型胶原的分泌,RT-PCR检测TGF-β1mRNA表达。结果与低糖组比较,高糖培养的HKC中α-SMA、p-JAK2、p-STAT1和p-STAT3表达明显上调;E-Cadherin表达明显下调;TGF-β1mRNA表达增加;上清液中TGF-β1、Ⅰ型胶原增加。AG490明显抑制α-SMA表达升高,减轻E-Cadherin表达;降低TGF-β1mRNA表达及TGF-β1、Ⅰ型胶原的分泌。结论JAK/STAT信号途径可能参与高糖诱导HKC转分化。     

High glucose increases phosphocofilin via phosphorylation of LIM kinase due to Rho/Rho kinase activation in cultured pig proximal tubular epithelial cells

In proximal tubular epithelial cells (PTECs), depolymerization of actin by cofilin plays a crucial role in maintaining polarity and function. Cofilin is inactivated when phosphorylated by p-Lin-11/Isl-1/Mec-3 kinase (LIMK) to give p-cofilin. LIMK is phosphorylated by phosphorylated p21-activated kinase (PAK), a downstream signal of phosphoinositide 3-kinase (PI3K), or by Rho kinase (ROCK), and is dephosphorylated by slingshot (SSH). However, in PTECs the signaling pathways regulating phosphorylation and dephosphorylation of cofilin, and the influence of high glucose (HG) on these pathways remain to be elucidated. Here, we show that HG in cultured porcine PTECs (LLC-PK1) increases p-cofilin and p-LIMK1 beyond 6h and that the simultaneous presence of phlorizin reverses the increase. HG did not influence the levels of PI3K-p85, downstream signals to SSH1 and p-PAK1, and mRNA of cofilin, LIMK1 and SSH1. On the other hand, wortmannin and LY294002 markedly increased p-cofilin and p-LIMK1 without influencing on the level of SSH1 protein. HG-activated RhoA and ROCK2 beyond 3h, and phlorizin attenuated this activation. GF109203X inhibited HG-induced increase in membranous RhoA and ROCK2, and phorbol ester increased these proteins. Y27632 (a ROCK inhibitor) reversed HG-induced increases of p-cofilin and p-LIMK1. We conclude that HG increases p-cofilin by phosphorylating LIMK1 through activation of Rho/Rho kinase, probably due to diacylglycerol-sensitive PKC activation resulting from increased glucose influx. HG did not alter PI3K or its downstream signals, even though PI3K has a physiological role in maintaining the cofilin level by activating SSH1.     

高糖环境下近端肾小管上皮细胞c—fos／c—jun的表达

目的：研究高糖作用下近端肾小管上皮细胞cofos,c-jun的表达,探讨c-Fos/c-Jun异二聚体（活化蛋白－1,AP－1）在介导高糖致近端肾小管细胞过度合成细胞外基质（ECM）中的作用。方法：有杉LLC－PK1细胞株,将细胞分成正常对照组（NG组,5．5mmol/L D-葡萄糖）及高糖组（HG组,25mmol/L D－葡萄糖）;c-fos,c-jun mRNA检测运用半定量RT－PCR法,蛋白检测采用细胞免疫化学法。结果：HG组c-fos,c-jun mRNA分别在1、2h出现升高,并分别在12、24h达最高峰;NG组上述基因的表达均无显著改变;与NG组相比,HG组c-fos,c-jun mRNA分别升高1．63倍,1．67倍;HG组c-Fos蛋白水平无明显变化,c-Jun蛋白则显著升高达45％。结论：高糖能促进近端肾小管细胞c-fos,c-jun的表达,并可能通过AP－1（c-Fos/c-Jun)的形成增加介导高糖促进ECM表达的致病作用。     

Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells

Aims/hypothesis Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy.Methods Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca²⁺ was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay.Results Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3±0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1±0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca²⁺] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-. Pre-treatment with pertussis toxin abolished PKC-. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide.Conclusions/interpretation This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.Abbreviations Akt/PKB protein kinase B - DTT dithiothreitol - ERK extracellular signal-regulated kinase - GPCR G-protein coupled receptor - MAPK mitogen-activated protein kinase - OK opossum kidney - PI3-K phosphoinositide 3-kinase - PKC protein kinase C - PMA phorbol myristate acetate - PMSF phenylmethansulfonylfluoride - PTC proximal tubular cells - PTX pertussis toxin     

Endocytosis provides a major alternative pathway for lysosomal biogenesis in kidney proximal tubular cells

Recruitment of acid hydrolases to lysosomes generally occurs by intracellular sorting based on recognition of a common mannose 6-phosphate signal in the transGolgi network and selective transport to late endosomes/lysosomes. Here we provide evidence for an alternative, efficient secretion-recapture pathway mediated by megalin and exemplified by cathepsin B in kidney proximal convoluted tubules (PCT). We found that in mouse kidneys with defective megalin expression [megalin knockout (KO)] or apical PCT trafficking (ClC-5 KO), the (pro)cathepsin B mRNA level was essentially preserved, but the protein content was greatly decreased and the enzyme was excreted in the urine as mannose 6-phosphate-devoid species. In polarized PCT-derived cells, purified cathepsin B was avidly and selectively taken up at the apical membrane, and uptake was abolished by the megalin competitor, receptor-associated protein. Direct interaction of cathepsin B with megalin was demonstrated by surface plasmon resonance. Procathepsin B was detected in normal mouse serum. Purified cathepsin B injected into mice was efficiently taken up by kidneys (approximately 10% of injection) and targeted to lysosomes where it remained active, as shown by autoradiography and subcellular fractionation. A single cathepsin B injection into cathepsin B KO mice could reconstitute full lysosomal enzyme activity in the kidneys. These findings demonstrate a pathway whereby circulating lysosomal enzymes are continuously filtered in glomeruli, reabsorbed by megalin-mediated endocytosis, and transferred into lysosomes to exert their function, providing a major source of enzymes to PCT. These results also extend the significance of megalin in PCT and have several physiopathological and clinical implications.     

Hyperglycemia induces PAI-1 gene expression in adipose tissue by activation of the hexosamine biosynthetic pathway.

We examined whether acute in vivo increases in either plasma glucose or insulin concentrations stimulate PAI-1 gene expression in fat tissue. We studied chronically catheterized unstressed and awake, lean (approximately 300 g, n=12) and obese (approximately 450 g, n=12) Sprague-Dawley rats. Hyperglycemia (approximately 18mM) was induced for 3 h by glucose infusion during a pancreatic clamp (somatostatin inhibited endogenous insulin secretion). Compared with equivalent saline infusion, hyperglycemia induced a 6-7 fold increase in PAI-1 gene expression in both lean and obese rats (P<0.001). When the rate of cellular glucose uptake was matched during a euglycemic hyperinsulinemic (approximately 60 microU/ml) clamp, PAI-1 gene expression in both obese and lean rats was proportionately and significantly increased (P<0.001). We further examined whether induction of the hexosamine biosynthetic pathway would mimic the effects of hyperglycemia and hyperinsulinemia on PAI-1 gene expression. Indeed, infusion of glucosamine (GlcN, 30 micromol/kg/min), induced a approximately 3-4 fold increase (P<0.01) in PAI-1 gene expression in both lean and obese animals. While obese rats had a four times greater fat mass then the lean rats, PAI-1 gene expression remained significantly higher when expressed as per gram fat. Our results support the hypothesis that increased glucose uptake induces PAI-1 gene expression in adipose tissue, probably through the activation of the hexosamine biosynthetic pathway. These findings may account for some of the fibrinolytic alterations seen in obese type 2 diabetic humans.     

Developmental expression of the angiotensinogen gene in rat embryos

Angiotensin II is a potent octapeptide vasoconstrictor and regulator of cardiovascular and electrolyte homeostasis. Angiotensinogen, the protein precursor of angiotensin II, is synthesized by the liver and many other organs of the adult rat. To determine whether angiotensin may be present in early fetal development we analyzed rat embryonic tissues (chorionic membranes, head, and body) for the expression of the angiotensinogen gene during days 11-21 of embryogenesis. Angiotensinogen mRNA was detected at low levels in embryo bodies and yolk sac placenta from day 11 of gestation. An initial rise in the level was noted on day 13, reaching a plateau from day 17 of gestation to birth. Angiotensinogen mRNA levels of the embryonic head were about 10-fold less than those of the body on days 17-19 and increased to levels similar to those of the body on days 20-21. Angiotensinogen mRNA levels of the yolk sac placenta were about 20-fold higher than those in the embryonic body, but no angiotensinogen mRNA was detected in the chorioallantoic placenta. Angiotensinogen mRNA from both embryos and yolk sac placenta was larger by about 200 bases than the mRNA obtained from adult rat liver; this was shown to be a consequence of both the utilization of more distal polyadenylation sites and a longer poly(A) tract. These observations suggest the possibility of a biological function for angiotensinogen in the early development of the rat, and that polyadenylation site selection may alter the functional expression of the angiotensinogen gene in a developmentally specific manner.     

Aldosterone stimulates collagen gene expression and synthesis via activation of ERK1/2 in rat renal fibroblasts

Recently, we demonstrated that in rats treated chronically with aldosterone and salt, severe tubulointerstitial fibrosis is associated with the activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/2). Here, we investigated whether aldosterone stimulates collagen synthesis via ERK1/2-dependent pathways in cultured rat renal fibroblasts. Gene expression of mineralocorticoid receptor (MR) and types I, II, III, and IV collagen was measured by real-time polymerase chain reaction (PCR). MR protein expression and ERK1/2 activity were evaluated by Western blotting analysis with anti-MR and anti-phospho-ERK1/2 antibodies, respectively. Collagen synthesis was determined by [3H]-proline incorporation. Significant levels of MR mRNA and protein expression were observed in rat renal fibroblasts. Treatment with aldosterone (0.1 to 10 nmol/L) increased ERK1/2 phosphorylation in a concentration-dependent manner with a peak at 5 minutes. Aldosterone (10 nmol/L) also increased the mRNA levels of types I, III, and IV collagen at 36 hours but had no effect on the type II collagen mRNA level. [3H]-proline incorporation was significantly increased by aldosterone in both the medium and cell layer at 48 hours. Aldosterone-induced ERK1/2 phosphorylation was markedly attenuated by pretreatment with eplerenone (10 micromol/L), a selective MR antagonist, or PD98059 (10 micromol/L), a specific inhibitor of MAPK kinase/ERK kinase, which is the upstream activator of ERK1/2. In addition, both eplerenone and PD98059 prevented the aldosterone-induced increases in types I, III, and IV collagen mRNA and [3H]-proline incorporation. These results suggest that aldosterone stimulates collagen gene expression and synthesis via MR-mediated ERK1/2 activation in renal fibroblasts, which may contribute to the progression of aldosterone-induced tubulointerstitial fibrosis.     

High glucose reduces albumin uptake in cultured proximal tubular cells (LLC-PK1)

In this study, we clarify that high glucose inhibits albumin uptake in cultured LLC-PK1 cells. LLC-PK1 cells cultured for 6 days with 5.5-27.8 mM D-glucose were challenged by fluorescein isothiocyanate (FITC)-conjugated human albumin (HA). FITC-HA binding and uptake were inhibited by >5.5mM glucose (5.5 mM > (P < 0.01) 11.0 mM > (P < 0.05) 16.7 mM approximately= 27.8 mM). Analysis of FITC-HA binding and uptake at 5.5 and 16.7 mM D-glucose (high glucose, HG) showed decreased affinity (K(m) for binding: 35.5 mg/l versus 52.6 mg/l, K(m) for uptake; 41.3 mg/l versus 55.6 mg/l) and maximal velocity (B(max)--0.33 microg versus 0.27 microg/30 min/mg protein; U(max)--4.40 microg versus 3.48 microg/60 min/mg protein) at HG. A comparison of the time courses of FITC-HA binding and uptake at 5.5 mM glucose and at HG showed that HG suppressed them beyond 15 min (P < 0.005-0.001). Phlorizin (>0.25 mM) completely reversed the HG-induced inhibition of FITC-HA binding and uptake. High glucose decreased mRNA of GLUT-1 and SGLT-1, but did not influence that of SGLT-2. The simultaneous presence of Vitamin E (10(-6)M), Vitamin C (10(-6)M) and reduced glutathione (0.25 mM) reversed the suppressed FITC-HA binding and uptake by HG, while any one or two of these molecules, and various inhibitors of advanced glycation end products, failed to do so. In conclusion, a high glucose milieu causes inhibition of albumin binding and uptake in proximal tubular cells by increasing metabolic oxidative stress through excessive glucose flux via the sodium glucose transporter.     

去甲斑蝥素不依赖钙调蛋白磷酸酶信号通路抑制高糖刺激肾小管上皮细胞细胞外基质的表达

目的:在证实去甲斑蝥素(norcantharidin,NCTD)能减轻糖尿病肾病(DN)大鼠肾间质纤维化和抑制高糖刺激的肾小管上皮细胞细胞外基质表达的基础上,观察NCTD对高糖刺激的肾小管上皮细胞钙调蛋白磷酸酶(calcineurin,CaN)通路的影响,探讨NCTD抗DN肾小管间质纤维化与其抑制CaN的关系。方法:常规培养人肾小管上皮细胞(HK-2),转染CaN siRNA,细胞分五组:(1)正常糖组(D-glucose5.5mmol/L);(2)高糖组(HG,D-glucose30mmol/L);(3)高糖+CaN siRNA组;(4)高糖+CaN siRNA+NCTD(5mg/L)组;(5)高糖+NCTD(5mg/L)组。采用Western-blot、免疫荧光和实时定量PCR,观察NCDT对HK-2细胞CaN/NFAT通路的影响,明确CaN siRNA的干扰效果。采用Western blot,检测NCTD对转染CaN siRNA后的HK-2细胞纤维连接蛋白(FN),胶原蛋白IV(Collagen IV,Col IV)及转化生长因子β1(TGF-β1)蛋白表达的影响。结果:高糖可促进HK-2细胞CaNmRNA及蛋白的表达,NCTD可在基因及蛋白水平抑制CaN的表达(P0.05)。免疫荧光发现CaN下游活化T细胞核因子(NFATc)在正常对照组中存在于胞质,高糖刺激后细胞核内开始表达,高糖刺激30min后发生明显的核转位,NCTD能在一定程度上抑制核转位的发生,并能减少高糖刺激后核内NFATc蛋白的表达。转染CaN siRNA后,高糖刺激后HK-2细胞中CaN mRNA以及蛋白表达均降低,而FN,Col IV以及TGF-β1蛋白水平表达都明显增强(P0.05)。NCTD可抑制转染CaN siRNA后高糖刺激的HK-2细胞FN,Col IV和TGF-β1的表达。结论:NCTD能下调肾小管上皮细胞CaN表达,阻断CaN/NFATc信号通路;但NCTD抑制高糖刺激后肾小管上皮细胞细胞外基质的表达,与其阻断CaN/NFATc信号通路无关。     

脂联素对高糖介导的大鼠近端肾小管上皮细胞单核趋化蛋白-1表达的影响

目的 体外实验研究脂联素对高糖刺激下的大鼠近端肾小管上皮细胞（NRK-52E）单核趋化蛋白-1（MCP-1）mRNA及蛋白表达的影响.方法 用含不同浓度葡萄糖的DMEM培养基体外培养NRK-52E细胞,分5组（每组4个样本,此实验重复4次）：A组：含5 mmol/L葡萄糖培养基对照组;B组：含30 mmol/L葡萄糖培养基组;C组：含30 nmol/L葡萄糖培养基＋1 mg/L脂联素组;D组：含30 mmol/L葡萄糖培养基＋5 mg/L脂联素组;E组：含30 mmol/L葡萄糖培养基＋10 mg/L脂联素组.以逆转录-聚合酶链反应（RT-PCR）、Western blot法榆测比较各组细胞MCP-1 mRNA及蛋白表达的变化.组间比较采用t检验,多组间比较采用方差分析.结果 A组细胞MCP-1 mRNA表达量为0.247±0.005,B组为0.691±0.009,显著高于A组（t=72.03,P＜0.01）;C组为0.425±0.013,显著高于A组（t=46.31,P＜0.05）;D组为0.307±0.012,与A组相近（t=73.24,P＞0.05）;E组为0.253±0.011,与A组无差异.不同浓度脂联素各组间比较差异亦具有统计学意义（F=37.15,P＜0.05）.A组MCP-1蛋白表达量为10.25±0.03,B组为58.47±0.02,显著高于A组（t=35.21,P＜0.01）;C组为35.86±0.05,较B组显著下降（t=48.26.21,P＜0.05）;D组为25.63±0.06,较B组显著下降（t=32.34,P＜0.01）;E组为21.53±0.03,较B组显著下降（t=42.26,P＜0.05）,但高于A组（t=64.28,P＜0.01）.不同浓度脂联素各组间比较差异亦具有统计学意义（F=53.15,P＜0.05）.结论 脂联素可呈剂量依赖性地抑制高糖环境下大鼠近端肾小管上皮细胞MCP-1 mRNA及蛋白的高表达.     

Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.