伊贝沙坦预防链脲佐菌素诱导糖尿病大鼠肾损伤的作用机制(英文)

目的:探讨血管紧张素Ⅱ受体拮抗剂伊贝沙坦(Irb)对链脲佐菌素(STZ)诱导的糖尿病大鼠的肾保护作用机制。方法:SD大鼠随机分为3组:正常对照组(N组,n=7),糖尿病肾病组(DN组,n=6)Irb治疗组(DNI组,n=7)。SD大鼠单侧肾切除后,腹腔注射STZ诱导糖尿病模型,观察治疗后第4, 8,12周的血糖(BG)、体重(BW)、尿白蛋白排泄、24小时尿总蛋白的改变,同时观察12周时肌酐清除率(Ccr)、肾重(KW)、肾脏肥大指数(KW/BW)、肾组织总蛋白含量(RTP)、肾小球面积(A_G)和体积(V_G)、肾小球毛细血管基底膜(GBM)厚度的改变。通过免疫组化观察肾组织结缔组织生长因子(CTGF)和转化生长因子-β_1(TGF-β_1)的表达。结果:DN组和DNI组间BG无差异(p>0.05)。Irb可显著抑制大鼠尿白蛋白、24小时尿总蛋白的排泄,抑制过度增高的Ccr(均为P<0.01)。而且Irb可显著抑制糖尿病大鼠KW、KW/BW、RTP、A_G和V_G的增加(P<0.05或P<0.01)。我们首次证实Irb能够显著抑制GBM的增厚和CTGF的表达(均为P<0.01)。另外,大鼠肾脏CTGF的表达与TGF-β_1的表达和肾小球体积呈显著正相关(均为P<0.01)。结论:早期应用Irb对糖尿病大鼠的肾保护作用可能与抑制肾脏肥大和肾脏CTGF表达有关。     

Renin-angiotensin system blockade prevents the increase in plasma transforming growth factor beta 1, and reduces proteinuria and kidney hypertrophy in the streptozotocin-diabetic rat.

INTRODUCTION: Combination therapy with angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) is used to improve renal outcome achieved by monotherapy in diabetic patients. In addition, interference with the renin-angiotensin system (RAS) reduced expression and excretion of transforming growth factor beta 1 (TGF-beta 1) in diabetic nephropathy. The aim of this study was to investigate the effects of interrupting the RAS by ACE inhibitor (ACE-I) or ARB monotherapy or by combination therapy on proteinuria, kidney hypertrophy and plasma TGF-beta 1 in diabetic rats. MATERIALS AND METHODS: Forty-one male Wistar rats were allocated to five groups: 1 = control rats, 2 = diabetic rats (streptozotocin [STZ] 55 mg/kg), 3 = diabetic rats as above receiving enalapril (20 mg/kg/day), 4 = diabetic rats receiving losartan (80 mg/kg/day), 5 = diabetic rats receiving both losartan and enalapril. The study lasted 60 days. RESULTS: Urinary protein excretion, kidney weight, serum ACE activity and plasma TGF-beta1 increased significantly in untreated diabetic rats compared with controls. Administration of losartan, enalapril, or both for 60 days prevented these changes. Furthermore, combined therapy for 30 days normalised urinary protein excretion, while monotherapy did not. Losartan inhibited serum ACE activity both in vivo and in vitro. Plasma TGF-beta 1 levels were positively correlated with blood glucose levels (r=0.4059) and with urinary protein excretion (r=0.3558). CONCLUSIONS: Combination therapy with losartan and enalapril was more effective than monotherapy with either drug in achieving an early antiproteinuric response. Long-term treatment with losartan was as effective as the combined treatment, possibly due to a dual inhibitory effect on the RAS. The antiproteinuric effect may be related, in part, to reduced TGF-beta 1.     

Prevention of early renal injury by mycophenolate mofetil and its mechanism in experimental diabetes

Previously it was shown that treatment with mycophenolate mofetil (MMF) attenuated renal inflammation and glomerular injury in a model of diabetes. However, the mechanism involved in the renoprotective effects of MMF in experimental diabetes has not been clearly delineated. Diabetes was induced by injection of streptozotocin (STZ) after uninephrectomy. Diabetic animals received no treatment or treatment with MMF (10 mg/kg once daily by gastric gavage) for 8 weeks, non-diabetic rats served as controls. Level of malondialdehyde (MDA) in renal tissue and urine as well as the activity of antioxidant enzymes (AOE) in renal tissue was determined. Renal injury was evaluated. Immunohistochemistry for ED-1 macrophages marker, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) was performed. Expression of transforming growth factor (TGF)-beta1 protein was determined by Western blotting analysis. Treatment with MMF had no effect on blood glucose level, but did prevent increased urinary albumin excretion and glomerular damage in diabetic rats. Oxidative stress was reduced by MMF treatment, as indicated by a reduction in MDA level in renal tissue and urine. Activity of AOE such as glutathione peroxidase (GSH-PX) was markedly elevated while superoxide dismutase (SOD) and catalase (CAT) were not changed by MMF treatment. In diabetic animals receiving no treatment, there were increases in ED-1-positive cells, ICAM-1 expression and MCP-1 expression in glomeruli and tubulointerstitium, which were effectively suppressed by MMF treatment. Western blotting analysis showed that the expression of TGF-beta1 protein was increased by 1.92-fold in renal tissue in diabetic rats, and MMF treatment significantly reduced the increased expression of TGF-beta1 protein by 45%. Our data suggest that MMF treatment ameliorates early renal injury via the inhibition of oxidative stress and overexpression of ICAM-1, MCP-1 and TGF-beta1 in renal tissue in diabetic rats.     

Effect of telmisartan on expression of protein kinase C-α in kidneys of diabetic mice

Aim： To investigate the effects of angiotensin receptor blocker （ARB） telmisartan on the expression and distribution of protein kinase C （PKC）-α in the kidneys of diabetic mice. Methods： Diabetic mice were induced with streptozotocin and a group of them were randomly selected for treatment with telmisartan. After 6 weeks, the expression and localization of PKC-α in the renal cortex, and the outer and inner medulla were assessed by immunohistochemistry and semiquantitative Western blotting. In addition, expressions of PKC-α, transforming growth factor- β1 （TGF-β1）, and vascular endothelial growth factor （VEGF） in glomeruli were measured by semiquantitative immunohistochemistry. Results： Diabetic and normal mice showed similar distributions of PKC-α in the kidneys. The expression of PKC-α was found in glomeruli, epithelial cells of proximal tubules, and medullary- collecting duct, while not in the medullary and cortical thick ascending limb, and was different in the epithelial cells of proximal tubules of diabetic nephropathy （DN） mice, PKC-α was mostly translocated from the basement membrane to the apical membrane, whereas it was largely translocated from the apical membrane to the basement membrane in epithelial cells of the inner medullary-collecting duct. Western blotting detected increased expression of PKC-α in the renal cortex and outer medulla, but not in the inner medulla of DN mice. Enhanced expressions of PKC-α, TGF-β1, and VEGF were shown in the glomeruli of DN mice, where PKC-α exhibited a correlation to VEGF, but no correlation to TGF-β1. ARB telmisartan attenuated alterations of PKC-α as mentioned earlier in the DN mice. Conclusion： Our findings suggest that PKC-α may play a role in the pathogenesis of DN, and that the nephroprotective effects of ARB telmisartan may be partly associated with its influence on PKC-α.     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

盐酸贝那普利对2型糖尿病大鼠肾脏VEGF表达的影响

目的探讨血管紧张素转换酶抑制剂（ACEI）——盐酸贝那普利对糖尿病大鼠肾脏VEGF表达的影响。方法采用长期高脂饮食加小剂量链脲菌素（STZ）一次性腹腔注射制作2型糖尿病大鼠模型,光镜及电镜行肾脏形态学检查;RT-PCR测定VEGF mRNA表达,VEGF免疫组化染色观察VEGF蛋白水平变化。结果2型糖尿病大鼠肾脏VEGF mRNA及其蛋白质表达明显高于正常对照组（P〈0.01）,同时大鼠肾脏出现糖尿病肾病的病理变化;ACEI盐酸贝那普利干预后,对照组肾组织VEGF mRNA及其蛋白质表达较模型组降低（P〈0.01）,同时肾脏病理变化也得到明显改善。结论盐酸贝那普利可能通过抑制肾脏VEGF表达,延缓糖尿病肾病的发生。     

Renoprotective effects of fenofibrate in diabetic rats are achieved by suppressing kidney plasminogen activator inhibitor-1

To investigate mechanisms of protective effects of fenofibrate on the diabetic kidney, male Wistar rats were divided into control, untreated diabetes, and fenofibrate-treated (32 mg kg(-1) d(-1), 8 weeks) diabetes groups. Diabetes induced by streptozotocin (25 mg/kg) and a high-fat diet was characterized by the disorders of plasma glucose and lipids. In untreated diabetic rats, there were increases in glomerular volume, matrix content, expressions of laminin and urinary albumin excretion. These nephropathies were associated with the upregulations of plasminogen activator inhibitor 1 (PAI-1) mRNA expression and its protein activity in the renal cortex, and a significant increase in transforming growth factor beta1 (TGF-beta1) expression. Treatment with fenofibrate suppressed the expression of PAI-I mRNA and its protein activity, and inhibited TGF-beta1 overexpression. It also partially reversed metabolic disorders and pathophysiologic changes associated with diabetic nephropathy. Our results indicate that fenofibrate delays the progression of diabetic nephropathy in rats to some extent. These renoprotective effects are likely to be achieved through suppression of PAI-1 and TGF-beta1 in the renal cortex, and consequently less extracellular matrix deposition.     

霉酚酸酯对大鼠糖尿病肾病及CD2相关蛋白表达的影响

目的研究霉酚酸酯（MMF）对大鼠糖尿病肾病（DN）及CD2相关蛋白（CD2AP）表达的影响。方法纯种雄性SD大鼠24只,分正常对照（A）组、DN模型（B）组和MMF干预DN（C）组。饲养8周后行有关血尿生化指标和光镜组织学、透射电镜、逆转录聚合酶链反应（RT-PCR）CD2AP mRNA表达检测。结果MMF明显减少DN大鼠24h尿蛋白的排泄,预防血肌酐的升高。组织学显示C组大鼠肾小球面积明显减少,系膜增宽及细胞外基质积聚明显改善。电镜下B组大鼠足细胞足突增宽,部分融合,肾小球毛细血管袢基膜增厚,系膜区增宽,系膜基质增多,C组大鼠以上病变都明显减轻。CD2AP mRNA表达在B组显著高于A组和C组。结论MMF具有治疗SD大鼠DN的作用,并且在转录水平逆转肾皮质CD2AP表达上调。     

Double-edged action of SOD mimetic in diabetic nephropathy

Oxidative stress plays an important role in the pathogenesis of diabetic complications, and we investigated the effect of superoxide dismutase (SOD) mimetic, tempol, in diabetic nephropathy. Streptozotocin-induced diabetic rats were treated with tempol from 2 weeks until 8 weeks. The expression of NADPH oxidase, catalase, and myeloperoxidase (MPO), superoxide dismutase activity, and production of peroxide and hypochlorite were evaluated. Tempol treatment prevented the increase in NADPH oxidase and peroxide production in the glomeruli of diabetic rat. Catalase was decreased without change in SOD activity, and MPO was enhanced in the kidney of diabetic rats. Tempol treatment stimulated SOD activity and increased the conversion of superoxide to hydrogen peroxide, and hydrogen peroxide on its hand was converted to hypochlorite by the increased MPO. The reduction of peroxide by tempol was followed by the decrease in TGF-beta and mesangial matrix expansion. However, tempol did not reduce hypochlorite or urinary protein excretion. In conclusion, tempol inhibited glomerular matrix expansion via suppression of peroxide production and TGF-beta, but it failed to reduce proteinuria, probably due to the increased hypochlorite production in diabetic nephropathy.     

灯盏花素对大鼠糖尿病模型肾组织TGF-β1与CTGF表达影响的实验研究

目的　探讨灯盏花素对大鼠糖尿病模型肾组织转化生长因子 β1(TGF β1)与结缔组织生长因子 (CTGF)表达的影响。方法　建立STZ诱导的单侧肾切除糖尿病模型 ,随机分 :对照组、模型组、灯盏花素给药组 (2 0mg·kg-1·d-1,灌胃 )。观察 8wk后大鼠体重、肾重、肾重 /体重、2 4h尿白蛋白排泄率 (AER)、肾小球面积 (AG)和容量 (VG)及系膜区面积 (AM)的变化 ,并检测肾组织与尿MDA含量及肾组织SOD、CAT、GSH PX活性 ,通过免疫组化检测肾小球TGF β1与CTGF蛋白表达。结果　灯盏花素给药对大鼠糖尿病模型血糖、体重无明显影响 ,可明显抑制肾重、肾重 /体重、AER、AG、VG 及AM 的增加 (P <0 0 5 ) ;对肾组织及尿MDA含量的增加有明显抑制作用 (P <0 0 5 ) ,并明显提高肾组织SOD、CAT及GSH PX活性 (P <0 0 5 ,P <0 0 1)。免疫组化半定量分析显示 ,与对照组相比糖尿病大鼠肾小球TGF β1及CTGF表达明显增加 (P <0 0 1) ,灯盏花素给药对其有明显抑制作用 (P <0 0 5 )。结论　灯盏花素对大鼠糖尿病模型肾脏有明显保护作用 ,其机制部分与抑制肾组织TGF β1与CTGF过高表达有关     

Application of an adenoviral vector encoding soluble transforming growth factor-beta type II receptor to the treatment of diabetic nephropathy in mice

1. In the present study, we examined the effects of inhibiting transforming growth factor (TGF)-beta in a mouse model of diabetic nephropathy. 2. An adenovirus harbouring the gene encoding soluble TGF-beta type II receptor (Ad.CAG-sTbetaRII), a competitive inhibitor of TGF-beta, was injected into hindlimb muscles (systemic delivery) of mice 5 weeks after the induction of diabetes with streptozotocin. The control group was injected with an adenovirus encoding the LacZ gene (Ad-LacZ). 3. Five weeks after administration, anti-TGF-beta gene therapy was found to have had no effect on renal function, albuminuria or glucose metabolism in mice with diabetic nephropathy. Nonetheless, this gene therapy did significantly reduce fibrosis in both glomeruli and renal tubules. These effects were accompanied by attenuation of the increased expression of alpha-smooth muscle actin normally seen in kidneys of diabetic mice and better preservation of glomerular cell numbers, although the thickness of the glomerular capillary basement membrane was unchanged. The plasma concentration of soluble TGF-beta type II receptor peaked on Day 7 after treatment, but was undetectable by Day 14. Moreover, a second treatment with Ad.CAG-sTbetaRII failed to prolong the interval of gene product expression in the blood. 4. The present anti-TGF-beta gene therapy showed a significant antifibrotic effect in a model of diabetic nephropathy, but failed to improve renal function. The inadequacy of the observed effect is likely due to the relatively short interval of gene product expression. This problem will have to be overcome if gene therapies for slowly progressing diseases, like diabetic nephropathy, are to be realised.     

Roles of coagulation pathway and factor Xa in the progression of diabetic nephropathy in db/db mice

The active type of coagulation factor X (factor Xa) activates various cell-types through protease-activated receptor 2 (PAR2). We previously reported that a factor Xa inhibitor could suppress Thy-1 nephritis. Considering that fibrin deposition is observed in diabetic nephropathy as well as in glomerulonephritis, this study examined the roles of the coagulation pathway and factor Xa in the development of diabetic nephropathy using type 2 diabetic model mice. Diabetic (db/db) and normoglycemic (m+/m+) mice were immunohistochemically evaluated for their expression/deposition of PAR2, transforming growth factor (TGF)-β, fibrin, extracellular matrix (ECM) proteins, and CD31 at week 20. Significantly greater numbers of PAR2-positive cells and larger amounts of fibronectin, and collagen IV depositions were observed in the glomeruli of db/db mice than those in m+/m+ mice. Next, expression of PAR2 versus deposition of collagen IV and fibronectin was compared between week 20 and week 30, and the number of PAR2-positive cells in the glomeruli decreased in contrast with the increased accumulation of ECM proteins. In an intervention study, fondaparinux, a factor Xa inhibitor, was subcutaneously administered for ten weeks from week 10 to 20. Fondaparinux treatment significantly suppressed urinary protein, glomerular hypertrophy, fibrin deposition, expression of connective tissue growth factor, and ECM proteins deposition together with CD31-positive capillaries. These results suggest that coagulation pathway and glomerular PAR2 expression are upregulated in the early phase of diabetes, together with the increase of profibrotic cytokines expression, ECM proteins deposition and CD-31-positive vessels. Factor Xa inhibition may ameliorate glomerular neoangiogenesis and ECM accumulation in diabetic nephropathy.     

Diabetes modulates the expression of glomerular kinin receptors

The localization of kinin receptors within the kidney implicates this system in the regulation of glomerular hemodynamics. We reported that diabetes alters the activity of the renal kallikrein-kinin system, and that these alterations contribute to the development of microvascular complications of diabetes. The present study examined the influence of diabetes on the expression of glomerular B1 and B2-kinin receptors, and assessed the cellular signaling of kinin receptor activation. Rats made diabetic with streptozocin (85 mg/kg), displayed plasma glucose levels in the range of 350-500 mg/dl. At 3, 7, and 21 days, B1 and B2-kinin receptor mRNA levels were measured in isolated glomeruli from control and diabetic rats by RT-PCR. Glomeruli revealed a differential pattern of expression between the two kinin receptors. The constitutively expressed B2-receptor was increased three-fold at day 3, but returned to normal levels at day 7; whereas, the inducible B1-receptor was maximally expressed (20-fold) at day 7 and remained elevated (10-fold) at day 21. To test whether the induction of kinin receptors by diabetes translates into increased responsiveness, we measured mitogen-activated protein kinase (MAPK) phosphorylation (p42, p44) in glomeruli isolated from control and diabetic rats stimulated with B1-receptor agonist (des-Arg9-bradykinin, 10(-8) M). A three-fold increase in phosphorylation of MAPK was observed in response to B1-receptor agonist challenge in glomeruli isolated form diabetic rats compared to controls. These findings demonstrate for the first time that glomerular kinin receptors are induced by diabetes, and provide a rationale to study the contribution of these receptors to the development of glomerular injury in diabetes.     

姜黄素抑制实验性糖尿病大鼠肾脏PDGF-B表达上调的作用

目的探讨姜黄素对实验性糖尿病大鼠肾脏血小板衍化生长因子-B（platelet-derived growthfactor-B,PDGF-B）表达上调的抑制作用。方法 36只大鼠随机分为正常对照组（A组）、糖尿病组（B组）及姜黄素治疗组（C组）,每组12只采用腹腔单剂量注射链脲佐菌素（streptozotocin,STZ）65mg/kg建立糖尿病大鼠模型。治疗组给予姜黄素30mg·kg-1·d-1腹腔注射。实验第3、6周3组分别宰杀6只大鼠,并检测24h尿白蛋白排泄率（UAER）、肌酐清除率（Ccr）及肾重/体重。采用免疫组织化学染色法检测肾皮质PDGF-B的蛋白表达。肾组织常规制备电镜切片。结果治疗组大鼠UAER、Ccr、肾重/体重均明显低于糖尿病组大鼠（P〈0.05）。免疫组织化学染色可见糖尿病大鼠肾组织PDGF-B表达明显增加（P〈0.01）,治疗组其表达受到明显抑制（P〈0.05或〈0.01）。电镜结果显示糖尿病组大鼠肾小球基底膜增厚,系膜区增宽,脏层细胞肥大,系膜细胞增生,肾脏病变广泛。治疗组其超微结构异常明显改善。结论姜黄素通过抑制糖尿病大鼠肾组织PDGF-B表达上调,减轻糖尿病大鼠肾脏病变。     

Activation of protective and damaging components of the cardiac renin-angiotensin system after myocardial infarction in experimental diabetes.

INTRODUCTION: Diabetes is associated with prolonged apoptotic cell death of cardiac myocytes and adverse remodelling after myocardial infarction (MI). Because the renin-angiotensin system (RAS) has a major role in the remodelling, we studied whether diabetes is associated with altered regulation of RAS after MI in rats. METHODS: Male Wistar rats were randomised to receive either streptozotocin (diabetic group) or citrate buffer (control group) intravenously. MI was produced four weeks later by ligating the left descending coronary artery. The rats were sacrificed 1, 4 and 12 weeks after the operation. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE 2), angiotensin type 1 and 2 receptors (AT(1)-receptor, AT(2)-receptor), and connective tissue growth factor (CTGF) mRNA expression were determined. RESULTS: The expression of both protective and damaging components of RAS increased after MI. However, myocardial ACE 2 and AT(2)-receptor messenger ribonucleic acid (mRNA) expression levels were significantly lower in diabetic compared to non-diabetic rats 1 week after MI. In contrast, AT(1)-receptor, ACE and CTGF mRNA levels were up-regulated in diabetic as compared with non-diabetic rats 12 weeks after MI. CONCLUSION: The activation of the protective components of RAS (ACE 2 and AT(2)-receptor) was blunted early after MI in diabetic rats, whereas the levels of ACE, AT(1)-receptor and CTGF mRNA leading to adverse effects on myocardium, were elevated in diabetic as compared with non-diabetic rats. This unbalanced activation of the RAS may influence the pathophysiology of myocardial injury in diabetes after MI.