肾脏局部醛固酮对糖尿病肾病大鼠肾小管间质转分化的作用

目的 探讨醛固酮对糖尿病肾病（DN）大鼠肾小管间质转分化的影响。 方法 采用Wistar大鼠腹腔注射链脲菌素（STZ,60 mg/kg）制备糖尿病模型,4周后尿蛋白>30 mg/d为DN模型成功（n=16）,随机分为DN组（n=8）和螺内酯组（SP组,n=8）,以另8只正常大鼠作为对照组（N组）。SP组给予螺内酯40 mg&#8226;kg-1&#8226;d-1,N组、DN组每日以等量清水灌胃。8周后处死大鼠,收集尿、血浆、肾组织检测24 h尿蛋白定量、血肌酐和肾脏病理变化;用放射免疫法检测血浆、肾组织醛固酮浓度;用免疫组化、Western印迹方法检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）蛋白的表达;用RT-PCR的方法检测E-cadherin、α-SMA mRNA的表达。 结果 与对照组比较,DN组尿蛋白排泄量、血肌酐均显著增加（均 P < 0.01）,肾组织E-cadherin蛋白和mRNA表达显著下调（均P < 0.01）,α-SMA蛋白和mRNA表达均显著上调（均P < 0.01）。DN组大鼠肾组织醛固酮显著升高[（24.71±5.30） ng/g比（16.38±2.85） ng/g,P < 0.01],与尿蛋白排泄量、血肌酐、α-SMA蛋白表达呈正相关（r = 0.737、0.574、0.688,均P < 0.05）,与E-cadherin蛋白表达呈负相关（r = －0.659,P < 0.01）。各组间血清醛固酮含量差异无统计学意义。与DN组比较,SP组大鼠尿蛋白排泄和血肌酐显著下降（均P < 0.01）,E-cadherin蛋白和mRNA表达上调（均P < 0.05）,而α-SMA蛋白和mRNA表达显著下调(均P < 0.01)。 结论 DN大鼠肾组织局部醛固酮参与了糖尿病肾病肾间质转分化,螺内酯可以阻断醛固酮与其受体结合,抑制肾小管间质转分化,从而起到肾脏保护作用。     

miR-210 agonist alleviates renal inflammatory response and fibrosis in diabetic kidney disease rats

Objective To investigate the protective effect and mechanism of microRNA-210 agonist (agomiR-210) on kidney in diabetic kidney disease (DKD) rats. Methods Thirty-six 5-week-old male SD rats were divided into normal control (NC) group, agomiR-NC control group, agomiR-210 control group, DKD model group, DKD+agomiR-NC group and DKD+agomiR-210 group, with 6 rats in each group. Diabetic rats were established by a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ), then were fed for 12 consecutive weeks to construct DKD model rats. During 2nd-4th week of continuous feeding, the rats in DKD+agomiR-210 group were injected with 20 nmol/kg agomiR-210 via tail vein twice a week. Blood glucose levels, 24 h urine albumin (Alb) and 24 h urine microalbumin (MAU) contents were measured regularly. At the end of the 12th week, the rats were sacrificed, and renal tissues were collected. The renal histopathological changes were assessed by HE, PAS and Masson staining methods. The mRNA and protein expression levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in renal tissues were detected by RT-qPCR and Western blot. The distributions and expressions of α-smooth muscle actin (α-SMA), typeⅠ collagen (Col-Ⅰ), type Ⅳ collagen (Col-Ⅳ) and fibronectin (FN) in renal tissues were detected by immunohistochemical method. The protein expression levels of phospho(p)-Smad3 and p-NF-κB p65 in renal tissues were detected by Western blot and immunohistochemical methods. Results Compared with DKD model group, the renal pathological damages in DKD+agomiR-210 group were improved, the blood glucose level, glycogen deposition and collagen accumulation were significantly decreased (all P＜0.05), the urinary excretions of Alb and MAU were significantly reduced (all P＜0.01), and the expressions of TNF-α, IL-1β, IL-6, α-SMA, Col-Ⅰ, Col-Ⅳ, FN, p-Smad3 and p-NF-κB p65 in renal tissues were significantly decreased (all P＜0.01). Conclusion AgomiR-210 can alleviate renal pathological changes and urinary Alb and MAU excretion in rats with DKD, which may be related to its inhibition of Smad3 and NF-κB activity.     

氧化应激介导糖尿病大鼠肾小管上皮细胞转分化及普罗布考的干预作用

目的 研究氧化应激在糖尿病肾病（DN）大鼠肾小管上皮细胞转分化中的作用,探讨抗氧化剂普罗布考对大鼠DN的肾脏保护作用。 方法 30只雄性SD大鼠随机分为正常对照组、DN组和普罗布考干预组（1％普罗布考饮食）,每组10只。分别于第3周、第8周及第12周检测24 h尿蛋白（UTP）;12周末检测各组大鼠血糖、血脂（胆固醇、三酰甘油）、Scr、肌酐清除率（Ccr）、肾脏组织匀浆液丙二醛（MDA）含量及谷胱甘肽过氧化物酶（GSH-Px）活性。肾组织病理切片行 HE和Masson染色;采用免疫组化和Western印迹检测肾组织核转录因子Sp1、α平滑肌肌动蛋白（α-SMA）及E钙黏蛋白（E-cadherin）表达。 结果 与正常对照组比较,DN组血糖、Scr、肾组织匀浆MDA和24 h UTP水平显著增高（均P < 0.01）,Ccr显著降低（P < 0.01）;肾组织肾小管损伤分数、α-SMA和 Sp1蛋白表达水平明显增高（均P < 0.01）;肾组织E-cadherin蛋白表达明显下调。肾组织MDA含量分别与α-SMA及Sp1蛋白表达呈正相关（r ＝ 0.896,P < 0.01;r ＝ 0.862,P < 0.01）,与E-cadherin蛋白表达呈负相关（r = -0.673, P < 0.01）。普罗布考干预组Scr、24 h UTP、肾组织MDA、肾小管损伤分数及肾组织α-SMA、 Sp1蛋白表达水平较DN组均明显降低（均P < 0.01）;Ccr和肾组织E-cadherin蛋白表达水平较DN组均明显增加（均P < 0.01）。 结论 氧化应激在DN大鼠肾小管上皮细胞转分化中起重要作用。普罗布考可能通过抗氧化、下调肾组织Sp1蛋白表达及抑制肾小管上皮细胞转分化延缓DN大鼠肾脏病变进展。     

益肾胶囊对糖尿病肾病大鼠肾组织JAK/STAT信号通路及α-SMA、FN的影响

目的:探讨益肾胶囊对糖尿病肾病(DN)大鼠24h尿蛋白定量、尿素氮(BUN)、血肌酐(Scr)、JAK/STAT及α-平滑肌激动蛋白(α-SMA)、纤维连接蛋白(FN)的影响及其机制。方法:SD大鼠55只,随机留取10只为正常对照组(N)。利用一次性腹腔注射链脲佐菌素的方法建立DN大鼠模型。造模成功大鼠按随机数字表法分为糖尿病肾病模型组(DN)、益肾胶囊治疗组(DN+Y)、氯沙坦钾治疗组(DN+L)、氯沙坦钾+益肾胶囊治疗组(DN+L+Y),每组10只,连续灌胃12周。于实验的12周检测24h尿蛋白定量;12周末处死大鼠,检测血糖、BUN、Scr;取大鼠肾组织做组织病理学观察;免疫组化法检测肾组织磷酸化JAK2(p-JAK2)、磷酸化STAT3(p-STAT3)、α-SMA、FN的表达。结果:实验12周末,与DN组比较,益肾胶囊治疗组24h尿蛋白定量、BUN、Scr明显降低(P<0.05),肾组织形态学改变较轻,p-JAK2、p-STAT3、α-SMA、FN的表达显著降低(P<0.05)。氯沙坦钾联合益肾胶囊治疗组与氯沙坦钾治疗组比较,差异有统计学意义(P<0.05)。结论:益肾胶囊可能通过抑制糖尿病肾病大鼠肾组织JAK/STAT信号通路的活化,下调α-SMA、FN的表达,延缓了糖尿病肾病的进展;益肾胶囊联合氯沙坦钾可更好地保护肾脏功能,提示二者联合使用有协同作用。     

Epithelial-to-Mesenchymal Transdifferentiation of Renal Tubular Epithelial Cell Mediated by Oxidative Stress and Intervention Effect of Probucol in Diabetic Nephropathy Rats

Objective: To elucidate the relationship of oxidative stress and specificity protein 1 (Sp1) in the process of epithelial-to-mesenchymal transdifferentiation (EMT) and also to investigate the molecular mechanism of protective effect of probucol on the pathogenesis of diabetic kidney disease (DKD). Methods: Thirty male Sprague–Dawley (SD) rats were randomly divided into control group, diabetic group, and diabetic group under probucol therapy (n = 10 per group). The biochemical indicators including 24-h urinary total protein (24-h UTP) excretion, blood glucose (BG), lipids [triglycerides (TGs), total cholesterol (TC)], serum creatinine (Scr), creatinine clearance rate (Ccr), kidney tissue malondialdehyde (MDA) level, and glutathione peroxidase (GSH-Px) activity were assessed in all groups. The renal pathological changes were evaluated by hematoxylin and eosin (HE) and Masson staining. The protein expression of Sp1, α-smooth muscle actin (α-SMA), and E-cadherin was also measured and analyzed by immunohistochemistry and Western blotting. Results: Compared with the control group, the BG, TC, Scr, 24-h UTP, and MDA level of renal tissue increased significantly and the Ccr reduced in the rats of diabetic group (all p < 0.01). The pathological scores and the expression of Sp1 and α-SMA in renal tissue were up-regulated (p < 0.01) and the expression of E-cadherin was down-regulated significantly in the diabetic animals (p < 0.01). In the diabetic animals treated with probucol, the renal injuries were alleviated (p < 0.01). Conclusions: Oxidative stress may play an important role in the EMT process of tubular epithelial cells. Probucol could ameliorate renal disease progression in this model of diabetic nephropathy, which might be due to an antioxidant action, down-regulation of Sp1 protein expression, and inhibition of renal tubular EMT.     

法舒地尔对糖尿病大鼠肾小管上皮细胞转分化的影响

目的 观察法舒地尔对糖尿病大鼠肾小管上皮细胞转分化的影响并探讨其机制。 方法 将实验性Wistar大鼠随机分为正常对照组、糖尿病组、法舒地尔治疗组。3个月后处死动物,PAS染色法观察肾小球病理变化,Masson染色法观察肾间质病理变化;免疫组化观察肾脏皮质ROCKⅠ、α平滑肌肌动蛋白（α-SMA）、E钙黏蛋白（E-cadherin）的变化和β连环蛋白（β-catenin)的细胞定位改变;Western印迹法观察肾脏皮质p-MYPT1、α-SMA、E-cadherin、胞膜β-catenin蛋白的变化;实时定量PCR法观察ROCKⅠ、E-cadherin和总β-catenin的mRNA变化。 结果 法舒地尔治疗可改善肾间质纤维化状况。与正常对照组相比,糖尿病组大鼠肾皮质内p-MYPT1、α-SMA蛋白表达增强（均P < 0.01）;E-cadherin、胞膜β-catenin的蛋白表达减弱（均P < 0.01）;ROCK1、总β-catenin mRNA表达增强（均P < 0.01）;E-cadherin mRNA表达减弱（P < 0.01）。与糖尿病组相比,治疗组p-MYPT1、α-SMA蛋白表达减弱 （均P < 0.01）;E-cadherin、胞膜β-catenin的蛋白表达增强（均P < 0.05）;ROCK1、β-catenin mRNA表达减弱（均P < 0.01）;E-cadherin mRNA表达增强（P < 0.01）。 结论 法舒地尔可能通过抑制Rho激酶活性减轻糖尿病大鼠肾小管上皮细胞转分化和肾间质纤维化,该作用可能与法舒地尔恢复肾小管上皮细胞的黏附特性与紧密连接复合体有关。     

加强对正常蛋白尿糖尿病肾脏疾病的认识

糖尿病肾脏疾病(DKD)是导致终末期肾脏病(ESRD)的主要原因,早期及时诊断对防治DKD进展非常重要。以往DKD临床诊断多注重尿白蛋白排泄率(UAER),并以此作为诊断与临床分期的重要依据。实际上许多糖尿病(DM)有时表现为无临床诊断价值的蛋白尿,而以肾功能减退为突出肾脏受累表现,即正常蛋白尿糖尿病肾脏疾病(NADKD),或称无蛋白尿糖尿病肾病(nonalbuminuric diabetic nephropathy),及糖尿病无蛋白尿肾功能不全(nonalbuminuric renal impairment in diabetes)。本文结合国内外研究现状,从流行病学、病理、临床特征、诊断及治疗等方面对NADKD进行了描述,以翼引起同行重视。     

Effects of endothelin receptor antagonists on the progression of diabetic nephropathy

BACKGROUND: Diabetic nephropathy is the leading cause of end-stage renal disease in European countries and is associated with an enhanced renal synthesis of endothelin (ET)-1. ETs are - beside their potent vasoconstrictor properties - very potent profibrotic acting paracrine hormones especially in the kidney. METHODS: We analyzed in rats with streptozotocin-induced diabetes the effects of an ETA-type (ETA) receptor antagonist (LU 135252) in comparison to a combined ETA/ETB receptor antagonist (LU 224332) on the expression of interstitial and glomerular collagen type I, III and IV as well as on fibronectin and laminin by quantitative immunohistochemistry using a computer-aided image analysis system. Global glomerular matrix deposition was analyzed after PAS staining. In addition to the morphometric examination of the kidneys, we also investigated GFR, urinary albumin and total protein excretion. The diabetic rats were treated for 36 weeks. RESULTS: Treatment with either LU 135252 or LU 224332 normalized the amount of PAS-positive material within the glomeruli. The expression of glomerular fibronectin and type IV collagen was increased 36 weeks after induction of diabetes. The overexpression of these two matrix proteins within the glomeruli of diabetic rats was completely abolished by both ET receptor antagonists, whereas protein excretion was only reduced by about 50% as compared to diabetic rats without treatment. CONCLUSION: The present study indicates that ETA receptor antagonists as well as combined ETA/ETB receptor antagonists reduce proteinuria and completely normalize the renal matrix protein expression in hyperglycemic rats with streptozotocin-induced diabetes. The antifibrotic effect seems to be mediated via the ETA receptor. ET receptor antagonists might be a new approach in the treatment of diabetic nephropathy.     

白芍总苷对糖尿病大鼠肾小管-间质细胞转分化的影响

目的探讨白芍总苷（TGP）对糖尿病大鼠肾小管-间质细胞转分化的影响。方法建立链脲佐菌素（STZ）诱导的大鼠糖尿病模型,将大鼠随机分对照组、模型组、TGP给药组（50、100、200mg·kg-1·d-1灌胃）,8w后观察肾小管-间质损伤指数（TII）的变化,应用免疫组化方法检测肾组织E-钙黏蛋白（E-cadherin,E-cad）、α平滑肌肌动蛋白（a-smooth muscleactin,α-SMA）以及波形蛋白（Vim—entin,Vh）的表达。结果TGP给药呈剂量依赖性降低糖尿病大鼠24h尿白蛋白排泄率。模型组TⅡ明显高于对照组,TGP给药组（100mg·kg-1·d-1与200mg·kg-1·d-1）TII明显低于模型组。模型组肾小管-间质E-cad表达明显低于对照组（P〈0．05）,TGP50、100、200mg·kg-1·d-1给药组E-cad表达明显高于模型组（P〈0．01）。模型组肾小管-间质α—SMA与Vim蛋白表达明显高于对照组（P〈0．叭）,TGP50、100、200mg·kg-1·d-1给药组肾小管-间质α-SMA与Vim蛋白表达明显低于模型组（P〈0．01）。结论TGP可降低肾小管-间质α—SMA和vim的表达,增加E—cad的表达,抑制肾小管-间质细胞转分化。     

Regression of nephropathy developed in diabetes by (Pro)renin receptor blockade

Activation of prorenin by (pro)renin receptor stimulates the tissue renin-angiotensin system and plays a significant role in the development of nephropathy in diabetic animals. This study examined whether (pro)renin receptor blockade inhibits the progression of nephropathy that has already developed in diabetic rats. Seventeen-week-old heminephrectomized streptozotocin-induced diabetic rats with an increased urinary protein excretion and a significant glomerulosclerosis had been treated for 12 wk with the (pro)renin receptor blocker (PRRB), angiotensin-converting enzyme inhibitor (ACEi), or vehicle peptide by using subcutaneously implanted osmotic minipumps. At the end of observation, in diabetic rats that were treated with vehicle, urinary protein excretion was progressively increased and a significant progression of glomerulosclerosis was observed. In diabetic rats that were treated with PRRB, however, no further increase in urinary protein excretion or glomerulosclerosis was observed, but 12-wk treatment with ACEi only attenuated further increases in urinary protein excretion and glomerulosclerosis. The enhanced expression of activated prorenin was observed in the kidneys of diabetic rats that were treated with vehicle, whereas it was markedly suppressed in the kidneys of diabetic rats that were treated with PRRB but not ACEi. These results suggest that (pro)renin receptor blockade does not only inhibit the progression of nephropathy but also reverses the glomerulosclerosis that has already developed in diabetic rats.     

肾安提取液对糖尿病肾脏病小鼠肾皮质糖基化终产物受体表达的影响

目的观察肾安提取液对糖尿病肾脏病（DKD）小鼠肾皮质糖基化终产物受体（RAGE）表达的影响。方法将54只小鼠随机分为6组：正常对照组（N组）、DKD模型组（D组）、厄贝沙坦组（EB组）、肾安高剂量组（SAG组）、肾安中剂量组（SAZ组）、肾安低剂量组（SAD组）,每组各9只。采用腹腔注射链脲佐菌素（STZ）建立DKD模型,EB组给予厄贝沙坦灌胃,肾安高、中、低剂量组分别给予高、中、低剂量。肾安提取液灌胃。4周后,检测各组相对肾质量、血糖、24h尿蛋白定量、血肌酐,观察各组肾皮质病理改变,实时荧光定量聚合酶链反应（RT-PCR）法检测肾皮质RAGEmRNA表达水平,免疫组织化学法检查肾皮质RAGE蛋白表达水平。结果D组小鼠相对肾质量、血糖、SCr、24h尿蛋白定量、RAGEmRNA和蛋白表达均明显高于N组（P〈0．01）,肾小球肥大、纤维化明显。药物干预4周后,小鼠肾脏病理改变减轻,EB组、SAG组、SAZ组相对肾质量、SCr、24h尿蛋白定量较D组显著降低（P〈0．05或P〈0．01）,SAD组24h尿蛋白定量较D组显著降低（P〈0．05）。EB组、SAG组、SAZ组和SAD组肾皮质RAGEmRNA和蛋白表达较D组显著降低（P〈0．05或P〈0．01）。结论肾安提取液能保护DKD小鼠肾组织,其机制可能与降低肾皮质RAGE表达有关。     

Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice

Diabetic nephropathy is characterized by progressive loss of renal function, persistent proteinuria, and relentless accumulation of extracellular matrix leading to glomerulosclerosis and interstitial fibrosis. This study investigated the potential effects of long-term expression of exogenous hepatocyte growth factor (HGF) on normal and diabetic kidneys. Intravenous injection of human HGF gene via naked plasmid vector resulted in abundant HGF protein specifically localized in renal glomeruli, despite an extremely low level of transgene mRNA in the kidney. In uninephrectomized mice made diabetic with streptozotocin, delivery of exogenous HGF gene ameliorated the progression of diabetic nephropathy. HGF attenuated urine albumin and total protein excretion in diabetic mice. Exogenous HGF also mitigated glomerular mesangial expansion, reduced fibronectin and type I collagen deposition, and prevented interstitial myofibroblast activation. In addition, HGF prevented kidney cells from apoptotic death in the glomeruli and tubulointerstitium. Moreover, expression of HGF inhibited renal expression of TGF-beta1 and reduced urine level of TGF-beta1 protein. Therefore, despite the effects of HGF on diabetic nephropathy being controversial, these observations suggest that supplementation of HGF is beneficial in ameliorating diabetic renal insufficiency in mice.     

福辛普利对糖尿病大鼠肾脏NADPH氧化酶p22phox亚基mRNA表达的影响

目的 观察福辛普利对链脲佐菌素(STZ)诱导的糖尿病大鼠肾脏还原型辅酶Ⅱ(NADPH)氧化酶p22phox 亚基mRNA表达及细胞外基质（ECM）聚积的影响，并探讨其可能机制。 方法 STZ诱导的糖尿病大鼠模型，随机分为糖尿病非治疗组（DM组）和福辛普利治疗组（DM+Fosin组，福辛普利10 mg·kg-1·d-1），疗程12周。RT-PCR法检测肾脏NADPH氧化酶p22phox mRNA的表达；免疫组织化学方法检测肾脏纤连蛋白（FN）表达；白明胶酶谱法检测肾脏基质金属蛋白酶9（MMP-9）的活性；并检测Scr、尿蛋白排泄量和肾质量指数等指标。 结果 实验第4 周，DM+Fosin组大鼠肾脏NADPH氧化酶p22phox mRNA表达较DM组减少45％（P ＜ 0.05）。第8周时，DM+Fosin组的肾小球和肾小管间质FN表达较DM组分别降低52.5％和42.9％（均P ＜ 0.05）；肾脏MMP-9的活性升高29.6％（P ＜ 0.05）。实验第12周时，DM+Fosin组大鼠Scr水平、尿蛋白量（24 h）和肾质量指数较DM组分别降低35.9％、50.2％和17.2％（均P ＜ 0.05）。DM+Fosin组和DM组血糖的差异无统计学意义（P ＞ 0.05）。 结论 福辛普利可延缓糖尿病肾病的发生和发展，其机制可能与通过抑制NADPH氧化酶p22phox亚基mRNA的表达有关。     

蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞自噬-溶酶体通路的影响

目的探讨蝉花菌丝联合海昆肾喜胶囊对糖尿病肾病(DN)肾小管上皮细胞自噬-溶酶体的影响与机制。 方法将24只大鼠腹腔单次注射链脲佐菌素(STZ)诱导SD大鼠高血糖模型后,随机分为模型组(DN)和治疗组(DN+蝉花菌丝联合海昆肾喜胶囊),用药24周;另外10只未诱导高血糖大鼠设为对照组。检测各组大鼠肾功能指标,取肾皮质行组织病理学检查,采用Western印迹检测大鼠分离肾小管的胶原蛋白-Ⅲ、α-平滑肌肌动蛋白(α-SMA)、转录因子EB(TFEB)、自噬泡标记蛋白LC3和自噬底物p62表达。将HK-2细胞分为牛血清白蛋白(Co-BSA)组、Co-BSA治疗组、糖基化终末产物-BSA(AGEs-BSA)组及AGEs-BSA-治疗组。Western印迹检测各组细胞p62、TFEB表达;以TFEB质粒转染HK-2细胞,通过RT-PCR检测过表达效率,观察各组HK-2细胞溶酶体红色荧光探针荧光强度,并检测各组细胞组织蛋白酶活性。 结果与对照组大鼠相比,模型组大鼠的血肌酐(Scr)、24 h尿蛋白水平增高、LC3、p62、胶原蛋白-Ⅲ和α-SMA表达明显增多,而肾小球滤过率(GFR)、TFEB表达明显减少。与模型组大鼠相比,治疗组大鼠的Scr、24 h尿蛋白均明显降低,GFR明显升高,LC3、p62、胶原蛋白-Ⅲ和α-SMA表达明显减少,TFEB表达明显增多。在HK-2细胞中,TFEB和p62的表达情况与在大鼠肾组织中相似。与Co-BSA组相比,AGEs-BSA组的探针荧光强度和组织蛋白酶活性明显减弱;过表达TFEB后,AGEs-BSA组的荧光强度和组织蛋白酶活性均明显增强。 结论蝉花菌丝联合海昆肾喜胶囊对糖尿病肾小管上皮细胞可能具有上调TFEB表达、调节自噬-溶酶体通路的作用。     

替米沙坦对老年糖尿病大鼠足细胞黏附的保护作用

目的 探讨替米沙坦对足细胞黏附的保护作用。 方法 用20月龄SD大鼠制备糖尿病模型，随机分为糖尿病组和替米沙坦治疗组，另设老年和6周龄对照组。于实验第4、8、12周末收集大鼠24 h尿，监测血压。12周后行组织病理学检查，检测尿白蛋白（24 h）和血、尿肌酐。以间接免疫荧光-hoechst 33342双染检测尿中足细胞数量。RT-PCR、免疫组化和Western印迹等方法检测肾小球中整合素α3、整合素连接激酶(ILK) mRNA和蛋白表达。 结果 随实验进程，老年糖尿病大鼠尿白蛋白、血压和尿中足细胞数量明显高于对照组(P <0.05)，且出现较为明显的病理学损害。肾小球整合素α3 mRNA及蛋白表达量减少， ILK表达明显增加(P < 0.05)。第12周，整合素α3蛋白表达与ILK表达呈负相关 (P < 0.05)。尿足细胞数量、尿白蛋白程度与ILK表达呈正相关 (P < 0.05)。替米沙坦明显减轻肾脏病理损害，增加整合素α3表达，降低ILK表达，减少尿足细胞数量。 结论 老年糖尿病大鼠足细胞的黏附能力下降，尿足细胞排泄增加。替米沙坦可增强足细胞黏附，对足细胞存活起重要作用。     

Diabetes-induced microvascular dysfunction in the hydronephrotic kidney: role of nitric oxide.

BACKGROUND: Renal hemodynamics in early diabetes are characterized by preglomerular and postglomerular vasodilation and increased glomerular capillary pressure, leading to hyperfiltration. Despite intensive research, the etiology of the renal vasodilation in diabetes remains a matter of debate. The present study investigated the controversial role of nitric oxide (NO) in the renal vasodilation in streptozotocin-induced diabetic rats. METHODS: In the renal microcirculation, basal tone and response to NO synthase blockade were studied using the in vivo hydronephrotic kidney technique. L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) was administered locally to avoid confounding by systemic blood pressure effects. The expression of endothelial NO synthase (eNOS) was investigated in total kidney by immunocytochemistry and in isolated renal vascular trees by Western blotting. Urinary excretion of nitrites/nitrates was measured. RESULTS: Diabetic rats demonstrated a significant basal vasodilation of all preglomerular and postglomerular vessels versus control rats. Vasoconstriction to L-NAME was significantly increased in diabetic vessels. After high-dose L-NAME, there was no difference in diameter between diabetic and control vessels, suggesting that the basal vasodilation is mediated by NO. Immunocytochemically, the expression of eNOS was mainly localized in the endothelium of preglomerular and postglomerular vessels and glomerular capillaries, and was increased in the diabetic kidneys. Immunoblots on isolated renal vascular trees revealed an up-regulation of eNOS protein expression in diabetic animals. The urinary excretion of nitrites/nitrates was elevated in diabetic rats. CONCLUSION: The present study suggests that an up-regulation of eNOS in the renal microvasculature, resulting in an increased basal generation of NO, is responsible for the intrarenal vasodilation characteristic of early diabetes.     

Pentoxifylline ameliorates renal tumor necrosis factor expression, sodium retention, and renal hypertrophy in diabetic rats

BACKGROUND/AIM: Diabetic nephropathy contributes substantially to cardiovascular morbidity and mortality associated with diabetes. Urinary tumor necrosis factor (TNF) excretion is increased during diabetes and serves as an important mediator of pathological changes during the initial stages of diabetic nephropathy, including sodium retention and renal hypertrophy. We tested the hypothesis that pentoxifylline (PTF), an agent that inhibits TNF synthesis, could prevent sodium retention and renal hypertrophy during diabetes. METHODS: Proximal and distal tubule TNF expression, urinary TNF excretion, sodium retention, and renal hypertrophy were examined in control, diabetic, and PTF-treated diabetic rats. RESULTS: TNF mRNA and protein levels were increased in proximal tubule cells isolated from diabetic rats compared to control rats. In contrast, TNF expression in distal tubule cells was not increased during diabetes. PTF prevented the increase in TNF mRNA and protein in proximal tubule cells during diabetes and reduced urinary TNF excretion. PTF therapy decreased whole animal sodium retention by enhancing urinary sodium excretion in diabetic rats. In addition, PTF reduced renal hypertrophy in diabetic rats. CONCLUSIONS: The proximal tubule is an important site of TNF production during diabetes and PTF is an effective therapy for preventing the pathological changes accompanying early diabetic nephropathy.     

Diltiazem attenuates oxidative stress in diabetic rats

Diabetic nephropathy is the main cause of end stage renal damage. Oxidative stress is involved in the etiology of diabetic nephropathy and intracellular calcium is reported to play a considerable role in the development of renal damage in the diabetic kidney. Calcium antagonism can slow the progression of renal impairment in diabetes. The present study was thus designed to examine the effect of a nondihydropyridine calcium channel blocker, diltiazem, on renal function, oxidative stress, and nitric oxide (NO) release in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by a single intraperitoneal injection of STZ (65 mg/kg) in rats. After 4 weeks of STZ injection, the rats were divided in to four groups: control rats, diabetic rats treated with saline, and two groups of diabetic rats treated with diltiazem (5 and 10 mg/kg, i.p, respectively) for 8 weeks starting from 4 weeks after STZ injection. Renal function was assessed by creatinine, blood urea nitrogen, creatinine clearance, and urea clearance. Oxidative stress was measured by renal malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase. We also measured renal nitrite levels. At the end of the 8 weeks, diabetic rats exhibited renal dysfunction as evidenced by reduced creatinine and urea clearance along with enhanced albumin excretion rate as compared with control rats. Biochemical analysis of kidneys revealed a marked increase in oxidative stress demonstrated by increased lipid peroxidation and decreased activities of key antioxidant enzymes, GSH, SOD, and catalase in diabetic rats. Release of NO also significantly higher in diabetic rats than controls. Chronic treatment with diltiazem in diabetic rats significantly attenuated both renal dysfunction and oxidative stress along with increased NO levels as compared with untreated diabetic rats. The kidneys of diabetic rats showed morphological changes such as hyaline casts, glomerular thickening, and moderate interstitial fibrosis and arteriolopathy, whereas diltiazem administration markedly prevented diabetic-induced renal morphological alterations. The present study suggests that oxidative stress/nitrosative stress is increased in the diabetic kidney and calcium channel blockage can prevent these changes. The results also suggest that in STZ-induced diabetic rats, the protective action of diltiazem might be mediated, at least in part, by its effect on tissue oxidant/antioxidant status.     

全反式维甲酸对糖尿病大鼠足细胞损伤的保护作用

目的探讨全反式维甲酸(ATRA)对糖尿病大鼠足细胞损伤的保护作用。方法24只链脲佐菌素(STZ)诱导的糖尿病大鼠模型,随机分为糖尿病组(DM)和治疗组(T)各12只,另以12只正常大鼠作为对照组(N)。T组给予ATRA 20 mg·kg-1·d-1灌胃。于第4、8周末检测各组大鼠尿蛋白定量(24 h)、Cer、肾重/体重、尿中足细胞以及肾脏病理改变。结果DM组尿蛋白定量(24 h)、Ccr、肾重/体重、尿中足细胞数[8周时,0．84(0．60～1．50)个/ml]均显著高于同期N组[0．03(0-0．15)个/m1],而肾小球足细胞数(8周时,11．27±2．15)显著低于同期N组(14．07±2．07),且足细胞足突增宽、融合。T组尿蛋白定量(24 h)、Cer、肾重/体重、尿中足细胞数较同期DM组显著降低[0．46(0．25～0．70)个/ml],且病理改变减轻。结论ATRA可以减少糖尿病大鼠尿足细胞的排泄、降低尿蛋白,对肾脏足细胞损伤具有保护作用。     

Salutary effect of pigment epithelium-derived factor in diabetic nephropathy: evidence for antifibrogenic activities

Diabetic nephropathy is a major complication of diabetes and a leading cause of end-stage renal diseases in the U.S. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor that has been extensively studied in diabetic retinopathy. Recently, we reported that PEDF is expressed at high levels in normal kidneys and that PEDF levels are decreased in kidneys of streptozotocin (STZ)-induced diabetic rats. In the present study, we injected STZ-diabetic rats with an adenovirus expressing PEDF (Ad-PEDF) to evaluate its effects in diabetes. The results showed that increased expression of PEDF in the kidney in response to Ad-PEDF delivery significantly alleviated microalbuminuria in early stages of diabetes. Administration of Ad-PEDF was found to prevent the overexpression of two major fibrogenic factors, transforming growth factor-beta (TGF-beta)1 and connective tissue growth factor (CTGF), and to significantly reduce the production of an extracellular matrix (ECM) protein in the diabetic kidney. Moreover, PEDF upregulated metalloproteinase-2 expression in diabetic kidney, which is responsible for ECM degradation. In cultured human mesangial cells, PEDF significantly inhibited the overexpression of TGF-beta1 and fibronectin induced by angiotensin II. PEDF also blocked the fibronectin production induced by TGF-beta1 through inhibition of Smad3 activation. These findings suggest that PEDF functions as an endogenous anti-TGF-beta and antifibrogenic factor in the kidney. A therapeutic potential of PEDF in diabetic nephropathy is supported by its downregulation in diabetes; its prevention of the overexpression of TGF-beta, CTGF, and ECM proteins in diabetic kidney; and its amelioration of proteinuria in diabetic rats following Ad-PEDF injection.