【作者】 汪凌昊；

【导师】 陶立坚；

【作者基本信息】 中南大学 ， 肾脏病学， 2012， 博士

【摘要】 研究背景糖尿病肾病(DN)是糖尿病的常见严重并发症之一,常发展成为终末期肾病(ESRD)。目前,临床治疗手段主要是控制血糖和抗高血压,但疗效有限,只能减慢糖尿病肾病向肾功能衰竭的进程。因此,必须针对糖尿病肾病的主要病理机制研发新的治疗药物。糖尿病肾病组织学损伤的主要特征是胶原、纤维连接蛋白等细胞外基质(ECM)成分在肾小球系膜和肾小管间质进行性沉积,最终导致不可逆的肾纤维化。近年来,不少研究着力于寻找能有效抑制ECM在组织中沉积而治疗糖尿病肾病的药物,取得了一些前景诱人的成果。AKF-PD是一种新合成的低分子量化合物。我们研究组最近报道,在体外培养的小鼠肾小球系膜细胞和大鼠肾小管上皮细胞中,AKF-PD能够抑制转化生长因子-β1(transforming growth factor-β1, TGF-β1)刺激后引起的ECM的产生。目的以糖尿病肾病db/db小鼠和MES13肾小球系膜细胞为实验对象,观察AKF-PD对肾纤维化的治疗效果,并深入揭示其作用机制,为研发治疗糖尿病肾病的抗纤维化新药提供实验证据。方法1.10周龄雄性db/db小鼠分为1个不给药模型组和3个AKF-PD给药组(剂量分别为每日125、500、1000mg/kg体重)；相同周龄雄性db/m小鼠作为正常对照组。称体重和测血糖后,AKF-PD溶于羧甲基纤维素钠溶液作灌胃给药,不给药db/db组和db/m组小鼠作等量羧甲基纤维素钠溶液灌胃,各组小鼠每天灌胃一次,持续12周。实验期间2周一次测血糖水平。实验12周后,小鼠分单只置于代谢笼,收集24h尿标本。用ELISA法测定尿白蛋白浓度。处死各组小鼠。称体重后,取双侧肾脏,称肾重和剪取肾皮质。制备肾皮质组织切片,作PAS染色。光镜下观察组织切片,按每只小鼠20个肾小球,分5级(0-4级)进行每个肾小球损伤程度的评分,并计算肾小球系膜基质扩展指数(GMI)。用ELISA法测定肾皮质组织匀浆TGF-β1蛋白水平。以β-actin为内参作标化,real-time PCR方法检测肾皮质组织Ⅰ型胶原、Ⅳ型胶原、纤维连接蛋白(FN)、金属蛋白酶组织抑制因子-1(TIMP-1)、α-平滑肌肌动蛋白(a-SMA)mRNA的相对丰度。用肾皮质组织裂解产物作SDS-PAGE,进行western blot检测。以a-tublin为内参标化,检测TIMP-1、α-SMA蛋白质的相对含量。2.细胞周期同步化处理的小鼠肾小球系膜细胞系(MES-13)细胞分为6个组：(1)NG组(正常浓度葡萄糖,,-5.6mmol/L);(2)HG组(高浓度葡萄糖,25mmol/L);(3) AKF-PD组(HG+400μg/mLAKF-PD);(4)PFD组(HG+400μg/mL吡非尼酮)；(5)Los组(HG+21μmol/L氯沙坦)；(6)OC组(渗透压对照组,NG+19.4mmol/L甘露醇)。各组细胞培养72h后,收集细胞和上清。用ELISA法测定细胞培养上清液的TGF-β1蛋白水平。以β-actin为内参,real-timePCR方法检测细胞Ⅰ型胶原、Ⅳ型胶原、TIMP-1、α-SMA mRNA的相对丰度。用细胞裂解产物作SDS-PAGE,进行western blot检测。以a-tublin为内参,检测TIMP-1、α-SMA蛋白质的相对含量。结果dbldb组小鼠的肾重明显大于db/m组小鼠(P<0.05)。与不给药组相比,AKF-PD给药组dbldb小鼠的肾重明显减轻(P<0.05),以500mg/kg剂量组效果最明显(P<0.01)。dbldb小鼠有严重的白蛋白尿,24h尿白蛋白排泄量达240.26±76.24μg,比db/m小鼠组升高8倍(P<0.01)。AKF-PD给药显著减轻dbldb小鼠的白蛋白尿(P<0.01),最大有效剂量为500mg/kg,可使24h尿白蛋白排泄量下降58.5%。从实验开始至实验结束,dbldb小鼠始终有严重的高血糖,血糖一般维持于30mmol/L以上。AKF-PD给药组与不给药组dbldb小鼠的血糖水平差异无显著性意义。与db/m小鼠相比,dbldb小鼠的肾小球系膜基质PAS阳性染色区明显扩大,肾小球系膜基质扩展指数(GMI)显著增加(P<0.01)。与不给药组dbldb小鼠比,AKF-PD给药组dbldb小鼠的肾小球系膜基质PAS阳性染色区明显缩小,GMI显著减低(P<0.05),其中,500mg/kg体重剂量组的GMI值减低约55.8%(P<0.01)。dbldb小鼠肾皮质的Ⅰ型胶原、Ⅳ型胶原和纤维连接蛋白的mRNA表达水平比db/m小鼠显著升高(P<0.01)。AKF-PD显著抑制三种细胞外基质成分mRNA的表达(P<0.05)dbldb小鼠肾皮质的TGF-β1mRNA和蛋白表达水平比db/m小鼠显著升高(P<0.01)。AKF-PD显著抑制dbldb小鼠肾皮质TGF-β1的表达(P<0.05)。dbldb小鼠肾皮质TIMP-1mRNA和蛋白表达水平显著高于db/m小鼠(P<0.01)。AKF-PD给药对dbldb小鼠肾皮质TIMP-1的高表达具有显著下调作用(P<0.01)。dbldb小鼠肾皮质α-SMA mRNA和蛋白的表达水平显著高于db/m小鼠(P<0.01)。AKF-PD给药明显抑制dbldb小鼠肾皮质α-SMA的表达(P<0.05),其最大有效剂量为500mg/kg。与NG组相比,HG组MES-13细胞的Ⅰ型胶原、Ⅳ型胶原mRNA表达水平显著增高(P<0.01)。与HG组相比,AKF-PD组、PFD组与氯沙坦组MES-13细胞的Ⅰ型胶原、Ⅳ型胶原mRNA表达水平明显降低(P<0.05)。且三种药物的作用相似。与NG组相比,HG组MES-13细胞的TGF-β1mRNA和蛋白表达水平显著上调(P<0.01)；与HG组相比,AKF-PD组、PFD组与氯沙坦组MES-13细胞的TGF-β1mRNA和蛋白表达水平明显降低(P<0.05)。而AKF-PD与PFD能使TGF-β1蛋白表达下调到接近NG组的水平,氯沙坦的这种作用较弱。与NG组相比,HG组MES-13细胞的TIMP-1mRNA和蛋白表达水平显著上调(P<0.01)；与HG组相比,AKF-PD组、PFD组与氯沙坦组MES-13细胞的TIMP-1mRNA和蛋白表达水平明显降低(P<0.01)。且三种药物的作用相似。与NG组相比,HG组MES-13细胞的α-SMA蛋白表达水平显著增高(P<0.01)；与HG组相比,AKF-PD组、PFD组与氯沙坦组MES-13细胞的α-SMA蛋白表达水平明显降低(P<0.05)。且三种药物的作用相似。结论：AKF-PD长期给药对db/db小鼠的糖尿病肾病有治疗作用,可显著减少糖尿病肾病db/db小鼠24小时尿白蛋白排泄量,减轻肾小球系膜基质扩展,抑制肾组织Ⅰ型胶原、Ⅳ型胶原、纤维连接蛋白等细胞外基质成分及TGF-β1、α-SMA、TIMP-1的表达。AKF-PD对db/db小鼠的高血糖无明显影响。AKF-PD能明显抑制高糖刺激下小鼠肾小球系膜细胞(MES-13)的Ⅰ型胶原、Ⅳ型胶原、TGF-β1、α-SMA、TIMP-1表达。AKF-PD治疗糖尿病肾病肾纤维化的作用机制与其抑制肾小球系膜细胞TGF-β1的产生,抑制肾小球系膜细胞向肌成纤维细胞表型的转分化,抑制细胞外基质降解调节分子TIMP-1表达,调节肾脏细胞外基质降解平衡有关。更多还原

【Abstract】 BackgroundDiabetic nephropathy (DN) is the common and serious complication of diabetes that often progresses to end-stage renal disease (ESRD). Available therapies only slow but do not halt the progression of renal dysfunction in diabetic nephropathy in clinical settings. It is therefore necessary to develop new therapeutic agents that target major pathological mechanisms of diabetic nephropathy. The histological injury of diabetic nephropathy is mainly characterized by the progressive accumulation of extracellular matrix (ECM) components such as collagen and fibronectin in the glomerular mesangium and tubulointerstitium, which ultimately results in the irreversible fibrosis of kidneys. Considerable efforts are being made to explore therapeutic agents effectively inhibiting ECM accumulation to treat diabetic nephropathy, and promising results have been obtained in recent years. Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD) is a novel low-molecular-weight pyridone agent. We recently reported that AKF-PD exerts a strong antifibrotic effect and inhibits TGF-β-stimulated production of ECM in mouse mesangial cells and rat proximal tubular epithelial cells in culture.ObjectiveThe present study was designed to evaluate the potential therapeutic efficacy of AKF-PD on kidney fibrosis and to obtain an insight into its underlying mechanisms of action in db/db mice with diabetic nephropathy and cultured murine mesangial-derived MES13cells exposed to high glucose, in order that this pyridone agent can be developed into a potent antifibrotic drug for the therapy of diabetic kidney disease. Method1. Male db/db mice and age-matched dblm mice were used for this study. Mice at10weeks of age were divided into five groups:a dblm mice group, a dbldb mice group, and three AKF-PD treatment groups in which dbldb mice were administered with AKF-PD (dissolved in CMCNa solution) at doses of125mg,500mg or1000mg·kg body weight-1·day-1respectively. The mice of dblm and dbldb groups were administered with CMCNa solution alone. The administration of AKF-PD or CMCNa solution was performed by gavage daily for a period of12weeks.Mice were individually housed in metabolic cages and the urine samples were collected over24hours. Levels of urinary albumin were quantified using a mouse albumin ELISA Kit.Mice were anesthetized with ether, and then blood samples were obtained from the tail vein. Levels of blood glucose were determined with an Automatic Analyzer.The ether-anesthetized mice were sacrificed at the end of12-wk treatment. The kidneys were rapidly dissected out, weighed and separate the cortices. Sections of renal tissues were prepared and stained with periodic acid-Schiff (PAS).20glomeruli were evaluated for each mouse. The degree of damage in each glomerulus was assessed using a semi-quantitative (grade0-4) scoring method. The glomerular matrix expansion index (GMI) was calculated according to the number of glomeruli in each grade of mesangial expansion.TGF-β1protein levels in homogenates of renal cortices were determined by using a TGF-β1ELISA kit.Total RNA was isolated from kidney tissues. The cDNA was synthesized from total RNA. RT-PCR primers for α1(I)collagen, α1(IV)collagen, fibronectin, TGF-(31, tissue inhibitors-1of metalloproteinases (TIMP-1), α-smooth muscle actin (a-SMA) and β-actin were ordered from Sangon Inc. Amplification reactions were performed in duplicate with SYBR Green PCR reagent kit. The relative abundance of mRNA was standardized with β-actin mRNA as the invariant control.The lysates of kidney cortex tissues were subjected to SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Following the blocking step, the electroblotted membranes were immunoblotted with the primary antibodies against a-SMA, TIMP1, a-tublin respectively. The membranes immunoblotted were incubated with horseradish peroxidase-labelled secondary antibodies. After washing, the membranes were exposed to X-ray film. The bands of resulting autoradiographs were quantified densitometrically using Bandscan software.2. The synchronized MES-13cells were assigned to six groups:(1) NG, medium containing normal glucose concentration (-5.6mmol/L);(2) HG, medium containing25mmol/L glucose;(3) AKF-PD, medium containing HG and400μg/mL AKF-PD;(4) PFD, medium containing HG and400μg/mL pirfenidone;(5) Los, medium containing HG and2μmol/L losartan;(6) OC, medium containing NG and19.4mmol/L mannitol as an osmotic control. The cells in all six groups were cultured for72h, and then cells and culture supernatants were collected for RT-PCR and Western blotting.TGF-β1protein levels in supernatants from MES-13cell cultures were determined by using a TGF-β1ELISA kit.Total RNA was isolated from MES-13cells. The cDNA was synthesized from total RNA. RT-PCR primers for α1(Ⅰ)collagen, al(IV)collagen, fibronectin, TGF-β1, tissue inhibitors-1of metalloproteinases (TIMP-1), cc-smooth muscle actin (a-SMA) and β-actin were ordered from Sangon Inc. Amplification reactions were performed in duplicate with SYBR Green PCR reagent kit. The relative abundance of mRNA was standardized with β-actin mRNA as the invariant control.The lysates of MES-13cells were subjected to SDS-PAGE and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Following the blocking step, the electroblotted membranes were immunoblotted with the primary antibodies against a-SMA, TIMP1, a-tublin respectively. The membranes immunoblotted were incubated with horseradish peroxidase-labelled secondary antibodies. After washing, the membranes were exposed to X-ray film. The bands of resulting autoradiographs were quantified densitometrically using Bandscan software.ResultsKidney weights of diabetic db/db mice were markedly greater than those of dblm mice(P<0.05). Treatment with AKF-PD reduced the kidney hypertrophy of diabetic dbldb mice, as indicated by decreased kidney weight and kidney/body weight ratio in treated dbldb mice compared with untreated dbldb mice. The diminished effect of AKF-PD on the renal hypertrophy was most obvious at a dosage of500mg/kg body weight per day(P<0.01).Diabetic db/db mice had heavy albuminuria. The urinary albumin excretion rate was higher by8-fold in dbldb mice than that in dblm mice group(P<0.01). The administration of AKF-PD significantly attenuated albuminuria of diabetic db/db mice and a dose of500mg/kg body weight was the peak-effective dose at which24-h urinary albumin excretion declined by58.5percent(P<0.01).The dbldb mice remained hyperglycemic throughout the experimental period, and their mean blood glucose levels were about5times as high as in dblm mice(P<0.01). No significant differences of blood glucose were observed between AKF-PD treated and untreated dbldb mice groups.In comparison with dblm mice, PAS-positive mesangial matrix areas were substantially enlarged and the glomerular mesangial expansion index (GMI) i.e., the ratio of mesangial matrix area divided by the tuft area, was significantly increased in dbldb mice(P<0.01). Compared with untreated dbldb mice group, the PAS-positive mesangial matrix areas and GMIs decreased obviously in AKF-PD-treated dbldb mice groups(P<0.05) and GMI was reduced by approximately55.8%at the dose of500mg/kg body weight(P<0.01).The mRNA levels of α1(Ⅰ)collagen, α1(Ⅳ)collagen and fibronectin in renal cortex of db/db mice were3-4times higher than those in dblm mice(P<0.01). The up-regulated expressions of the three ECM components at the transcriptional level were dose-dependently reduced in renal cortex of diabetic dbldb mice subjected to AKF-PD treatment(P<0.05).Diabetic db/db mice had the marked upregulation of expression TGF-β1mRNA and protein in renal cortex compared with dblm mice(P<0.01). AKF-PD treatment reduced significantly the enhanced expression of TGF-(31mRNA and protein in dbldb mice(P<0.05).Compared with dblm mice, expression of renal cortex TIMP-1was markedly upregulated in diabetic dbldb mice as indicated by real-time RT-PCR and western blotting(P<0.01). The upregulated renal expressions of TIMP-1mRNA and protein in dbldb mice were significantly inhibited by AKF-PD treatment(P<0.01).The mRNA and protein expressions of a-SMA in renal cortex of diabetic dbldb mice were markedly upregulated compared with dblm mice(P<0.01). AKF-PD administration to dbldb mice inhibited dramatically the upregulated expressions of a-SMA mRNA and protein in renal cortex(P<0.05), and the maximally effective doses were about500mg/kg body weight per day.The expression of α1(Ⅰ) and α1(Ⅳ) collagen mRNAs in HG group of MES-13cells was obviously increased compared with NG group(P<0.01). The addition of AKF-PD, PFD or losartan to culture media inhibited significantly the high glucose-induced upregulation of α1(Ⅰ) and α1(Ⅳ) collagen mRNA expression in mesangial cells(P<0.05), and there was no significant difference among the three agents.Each of AKF-PD, PFD and losartan can inhibit significantly the up-regulated expression of TGF-β1mRNA induced by high glucose in MES-13cells in vitro(P<0.01), and differences between the three agents were not statistically significant. AKF-PD, PFD and losartan also inhibit significantly the high glucose-stimulated upregulation of TGF-β1protein expression in the cultured mesangial cells(P<0.05). However, both AKF-PD and PFD can reverse the expression of TGF-β1protein approximately to the level of NG group, but this inhibitory effect of losartan seemed to be slightly weaker.Both mRNA and protein expression of TIMP-1by cultured mesangial cells were remarkably increased in HG group compared with the NG group (P<0.01). The levels of TIMP-1mRNA and protein in the AKF-PD-, PFD-and losartan-treatment groups were obviously lower than those in HG group(P<0.01). No significant difference in TIMP-1was observed among these three treatment groups.In comparison with NG group, the expression level of a-SMA protein in HG group was significantly higher(P<0.01). The addition of AKF-PD, PFD or losartan to the culture media significantly inhibited the expression of a-SMA protein by cultured mesangial cells under high glucose conditions(P<0.05). The differences in a-SMA protein expression among these three agents were not significant. ConclusionThis study demonstrates that long-term administration of AKF-PD attenuates renal hypertrophy, mesangial matrix expansion and albuminuria in the diabetic db/db mice despite continued hyperglycemia. Upregulated expressions of several ECM components, TIMP-1, TGF-β1and a-SMA at the transcriptional and protein levels are significantly inhibited by AKF-PD treatment in renal cortex of db/db mice and cultured mesangial cells under high glucose conditions. The results presented here suggest that AKF-PD is able to ameliorate the ECM production and degradation imbalance via the inhibition of TGF-β1pathway, resulting in attenuation of renal fibrosis associated with diabetic state. Therefore, use of AKF-PD as a potent antifibrotic agent holds great promise for the therapy of diabetic nephropathy.更多还原