4羟基壬烯醛在糖尿病大鼠肾脏中的表达及意义

目的:探讨糖尿病大鼠肾脏中4羟基壬烯醛(4-HNE)的表达情况及其作用机制。方法:链脲佐菌素(STZ)60 mg/kg一次性腹腔注射构建糖尿病大鼠模型组(D组),并设空白对照组(C组),分别检测大鼠4周、8周及12周的血肌酐、尿素氮、三酰甘油、胆固醇及24 h尿蛋白定量,光镜下观察肾脏病理改变,免疫组化及Western-blot观察各组大鼠肾脏4-HNE的表达情况。结果:D组大鼠4周、8周及12周的肌酐、尿素氮、三酰甘油及胆固醇逐渐升高,并均高于同一时间点的C组,结果差异有统计学意义(P<0.05),随病程的延长D组大鼠肾脏病理改变逐渐加重,基底膜逐渐增厚,细胞外基质逐渐增多,C组病理改变不明显。免疫组化及western-blot显示D组大鼠4周、8周及12周肾组织中4-HNE的表达逐渐升高,并明显高于C组,差异有统计学意义(P<0.05)。结论:糖尿病大鼠肾脏中4-HNE随着病程延长其表达量逐渐升高,提示脂质过氧化参与了糖尿病肾病的发生及进展。     

The effect of irbesartan on the expression of angiopoietin-like protein 2 in the kidneys of type2 diabetes rats

Objective To observe the effect of irbesartan on the expression of angiopoietin-like protein 2 (ANGPTL2) in the diabetic rats kidney and explore the underlying mechanism. Methods A total of sixty male SD rats were divided into normal control group (NC group, n=15) and experimental group (n=45) randomly. The experimental group was fed with high sugar-fat diet and given a low dose streptozocin（STZ 30 mg/kg）to establish type 2 diabetic model. Rats successfully induced diabetes were randomly divided into 2 groups: diabetes group (DM) and irbesartan group (DI). Weight, blood pressure, blood glucose, serum creatinine (Scr), blood urea nitrogen(BUN), 24 hour urinary albumin(UAL) and renal histomorphology were observed after drug intervention at the 4th, 8th and 12th weeks. The expression of ANGPTL2 in renal tissue were detected by immunohistochemistry, real-time PCR and Western blotting. Results The levels of Scr, BUN, TG, TC and UAL in group DM were higher than in group NC at the 4th, 8th and 12th week (all P＜0.05).Compared with that in group DM, above indexes were lower in group DI at the 4th, 8th and 12th week (all P＜0.05). The pathological changes of the kidney in group DM were more serious than that in group DI. The expression of ANGPTL2 in group DM was much higher than that in group NC at the 4th, 8th and 12th week (all P＜0.05), and irbesartan treatment inhibited the up-regulation of ANGPTL2 in group DI(all P＜0.05). Conclusion The expression of ANGPTL2 increases in T2DM rats kidney tissue with time and irbesartan can inhibit the up-regulation of ANGPTL2 in T2DM rats.     

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。     

Effects of irbesartan on the expression of hypoxia-induced factor 1α and connective tissue growth factor in the kidney of unilateral ureteral ligation operation model rats

Objective To investigate the expression of hypoxia-induced factor 1α (HIF-1α)and connective tissue growth factor (CTGF) in the kidneys of unilateral ureteral ligation operation (UUO)model rats and the effect of irbesartan on the expression. Methods Thirty healthy adult male SD rats were randomly divided into 3 groups: sham operation group (n＝10), UUO group (n＝10) and irbesartan group (n＝10, UUO rats treated with irbesartan by lavage 2 days before operation). The rats in sham group and UUO group were treated with equal normal saline by lavage. Renal function, histopathological changes, urinary protein of 24 hours in rats at week 2 were measured. In situ hybridization and Western blotting were applied to measure the expression of HIF-1α and CTGF. Results At week 2, the levels of BUN, Scr and the expressions of HIF-1α and CTGF were significantly increased in UUO group compared with those in sham group (all P＜0.01). There was significant positive correlation between HIF-1α mRNA and CTGF mRNA (r＝0.697, P＜0.01). Compared with UUO group, the levels of urine protein and Scr were significantly decreased [(103.44±8.76) mg/24 h vs (278.23±26.15) mg/24 h, P＜0.01; (109.15±3.93) μmol/L vs (185.04±13.45) μmol/L P＜0.01], and renal tubulointerstitial lesion area became smaller (0.28±0.02 vs 0.51±0.05, P＜0.01) in irbesartan group. The expression of HIF-1α mRNA and protein was also significantly decreased after the treatment of irbesartan (all P＜0.01). Conclusions The expressions of HIF-1α and CTGF in UUO rats increase significantly. Irbesartan can improve renal fibrosis through down-regulating the expression of HIF-1α and CTGF.     

Effects of bone morphogenetic protein 2 signal pathway on renal artery calcification in progression of diabetic nephropathy

Objective To explore the effects of renal artery calcification on the progression of diabetic nephropathy (DN), the activation and its role of bone morphogenetic protein 2(BMP2) signal pathway in renal artery of rats. Methods Sixty male SD rats were randomly divided into control group(CON group), DN group and DN with vascular calcification group (DN+VDN group). Rats of group DN and DN+VDN were fed with high sugar and fat diet and injected with streptozocin (STZ) into abdominal cavity to induce diabetes. After diabetic models were successfully made, rats of group DN+VDN were treated by vitamin D3 plus nicotine. The rats were sacrificed at 8th, 12th and 16th week respectively and the levels of renal function, blood glucose and 24 h urinary protein (24-h Upro) were measured. The pathologic changes to the renal artery were observed by von-Kossa staining and the calcium content was detected by calcium assay kit. The pathologic changes to the kidney were observed by HE. Immunohistochemistry was applied to detect the protein expression of BMP2/Smad1/Runx2/Osterix signal pathway in the renal artery and real-time PCR were applied to detect the mRNA expression levels of BMP2 and Runx2. Results The calcium content and the deposition of black granules in DN group were significantly higher than those in group CON and lower than DN+VDN group at each time point (P＜0.05). The renal function indices in group DN and group DN+VDN were gradually increased in 8th,12th and 16th weeks, and were higher than those in group CON (P＜0.05). Compared with that in DN group, although the level of BUN, Scr, Cys C and 24-h Upro in DN+VDN group rats were higher at different time point, the level of Cys C at each time point and the level of 24-h Upro in the 16th week showed significant differences (P＜0.05). The pathological damages of the kidney in group DN and DN+VDN showed a continual worsening trend and the pathological changes of the kidney in group DN+VDN were more serious than those in group DN. Furthermore, the levels of BMP2/Smad1/Runx2/Osterix signal protein and BMP2, Runx2 mRNA in DN rats were higher than those in CON group, lower than DN+VDN group at each time point (P＜0.05). Correlation analysis demonstrated that calcium content was positively correlated with serum BUN, Scr, Cys C, 24-h Upro and the expression of BMP2, Runx2 mRNA (r=0.835, 0.705, 0.829, 0.897, 0.641, 0.683, P＜0.01, respectively). Conclusion Renal artery calcification may participate in and promote the progression of DN, and the BMP2 signal pathway may be an important regulating factor in DN with renal artery calcification.     

辛夷挥发油对糖尿病大鼠肾组织中P-selectin mRNA表达的影响

目的：观察辛夷挥发油对糖尿病大鼠肾组织中P-selectin mRNA表达的影响,探讨其对糖尿病大鼠肾脏的保护作用及机制。方法：SD大鼠随机分为正常对照组、糖尿病对照组及辛夷挥发油大、中、小剂量治疗组。造模前、后及成模后第4、8、12周检测各组大鼠的体重、血糖、24h尿蛋白定量;第12周处死大鼠检测肾功能;光镜及电镜观察肾组织病理变化;Realtime PCR检测肾组织中P-selectin mRNA表达。结果：与正常对照组相比,糖尿病对照组血糖、肾重/体重、24h尿蛋白定量、血尿素氮、肾组织中P-selectin mRNA表达显著升高（P〈0.01）;血肌酐显著降低（P〈0.01）;病理改变较明显。辛夷挥发油各治疗组24h尿蛋白定量、肾组织中P-selectin mRNA表达较糖尿病对照组显著降低（P〈0.01）,病理改变亦较糖尿病对照组轻。结论：辛夷挥发油可能通过抑制糖尿病大鼠肾组织中P-selectin mRNA表达而对肾脏具有保护作用。     

Role of type Ⅲ sodium-dependent phosphate cotransporter PiT-1 in cardiac damages in uremic rats fed with high phosphate diet

Objective To evaluate the expression of type III sodium-dependent phosphate cotransporter (Pit-1) in cardiac damages in uremic rats fed with high phosphate diet. Methods Male SD rats were given an adenine and high phosphate diet as uremic rats (n＝18, uremic rats) or just high phosphate diet (NHP group, n＝6). After making uremic models, 18 rats were randomly divided into three groups as follow: uremic rats received high phosphate diet only (UHP group, n＝6); uremic rats received high phosphate diet and intraperitoneal phosphonoformic acid (PFA) 0.15 g·kg-1·d-1 (UHP＋PFA group, n＝6 ); uremic rats received high phosphates diet and intraperitoneal normal saline 0.15 g·kg-1·d-1 (UHP＋NS group, n＝6). At the end of 4th week and 8th week, serum Scr, Ca, P, and urine Ca, P were examined. At the end of the 8th week, the rats were sacrificed, and left ventricle weight, myocyte diameter, cardiac fibrosis were measured. The expression of PiT-1, TGF-β1, Cbfα-1, phosphorylation p38 mitogen-activated protein kinase (p-p38MAPK) and ERK (p-ERK) were determined by Western blotting. Results At the end of 4th week, Scr was higher in the uremic rats than that in the NHP rats. There was no significant difference of serum Ca, P, 24 h urinary P and Ca clearance among 4 groups (P＞0.05). At the end of 8th week, serum P was higher and Ca was lower in uremic groups than that in NHP group (P＜0.05). There was no significant difference of Scr, serum Ca, P, 24 h urinary P and Ca clearance among uremic groups (P＞0.05). Compared with those in NHP rats, the left ventricle weight, myocyte diameter, and the collagen volume fraction (%) increased in UHP group and UHP＋NS group (all P＜0.05), however, these parameters were ameliorated in UHP＋PFA group significantly (all P＜0.05). Western blotting showed that the expressions of PiT-1, TGF-β1, Cbfα-1, p-p38MAPK and p-ERK were higher in UHP group and UHP＋NS group than that in NHP group and they decreased significantly in UHP＋PFA group (all P＜0.05). Conclusions There are significant cardiac damages in uremic rats received high phosphate diet, companied with increased PiT-1, TGF-β1, Cbfα-1, p-p38MAPK, p-ERK expressions. The adverse effects are ameliorated by PiT-1 blocker, indicating that PiT-1 play a role in high phosphate diet induced cardiac damages in uremic rats.     

调节内质网应激对糖尿病小鼠肾脏SET7/9表达的影响

目的 探讨调节内质网应激对小鼠肾组织组蛋白甲基转移酶(HMT) SET7/9表达的影响及意义.方法 db/db小鼠按随机数字表法分为糖尿病肾病(DN)组和甜菜碱治疗(DN+B)组;db/m小鼠作为正常对照(NC)组,每组各18只.实验第4、8、12周末分别采用实时定量PCR和(或)Western印迹法测定小鼠肾组织SET7/9、葡萄糖调节蛋白(GRP)78、H3K4me2和单核细胞趋化蛋白1(MCP-1)表达水平;ELISA法测定24 h尿蛋白排泄率(UPER)和尿MCP-1浓度;全自动生化分析仪检测血糖(BG)、血肌酐、血尿素氮的动态改变;PAS染色观察肾脏病理改变.结果 与NC组比较,DN组BG、BUN、UPER、MCP-1均显著升高(均P＜0.05),且呈时间依赖性.DN组小鼠第4周末开始出现肾小球基底膜增厚、系膜细胞增生改变,第12周末出现明显系膜基质积聚.与NC组比较,DN组肾组织GRP78、SET7/9的mRNA和蛋白质表达水平均显著升高,H3K4me2蛋白水平也显著升高,且呈时间依赖效应.与DN组比较,甜菜碱治疗组小鼠肾小球病变明显减轻,GRP78、SET7/9的mRNA及蛋白表达水平显著降低,BG、BUN、UPER、MCP-1、H3K4me2水平显著降低(均P＜0.05).结论 内质网应激可能是介导糖尿病小鼠肾脏SET7/9表达的上游机制.     

乙酰肝素酶在糖尿病肾病大鼠蛋白尿发生中的作用

目的 观察糖尿病肾病大鼠肾组织乙酰肝素酶（HPA）的表达，探讨其在糖尿病肾病大鼠蛋白尿发生中的作用。 方法 SD健康大鼠被随机分为健康对照组（n = 6）、糖尿病6周组(DM6w, n = 10)和糖尿病12周组(DM12w, n = 10)，采用一次性腹腔注射链脲菌素（STZ）的方法建立糖尿病大鼠模型。分别于造模后6周和12周末测定各组大鼠相对肾质量、血糖、尿素氮、血肌酐、24 h尿量及尿蛋白量(24 h)等指标，并观察肾脏病理改变。RT-PCR和免疫组织化学法检测各组大鼠肾组织HPA mRNA和蛋白表达变化，并分析其与蛋白尿发生的相关性。 结果 （1）DM6w和DM12w组大鼠的相对肾质量、血糖、尿素氮、24 h尿量及尿蛋白量(24 h)与健康对照组相比明显升高, 差异均有统计学意义(P < 0.05或P < 0.01)。（2）DM6w和DM12w组大鼠HPA mRNA和蛋白表达比健康对照组均显著增高(P < 0.01)。（3）大鼠肾组织HPA mRNA和蛋白表达与尿蛋白量(24 h)之间均呈正相关 (r = 0.783，P < 0.01；r = 0.793，P < 0.01)。 结论 HPA在糖尿病肾病中的表达升高可能参与了糖尿病肾病蛋白尿的发生。     

肿瘤坏死因子相关的凋亡诱导配体系统和核因子κB在糖尿病大鼠肾脏中的表达

目的探讨肿瘤坏死因子相关的凋亡诱导配体（TRAIL）系统在糖尿病肾病发生发展中的作用。方法观察TRAIL及其死亡受体4（DR4）、诱骗受体2（DcR2）和核因子KB（NF-KB）在糖尿病大鼠不同时期肾脏中的表达及分析其与肾功能的关系。将80只Wistar大鼠随机分为对照组（NC组）和糖尿病组（DM组），一侧肾切除后，腹腔注射链脲佐菌素（STZ，55mg／kg）建立糖尿病大鼠模型。在第4、8、12、16周末，随机处死各组8只大鼠，收集血液、尿液和肾组织，检测血生化指标、尿蛋白量（24h）和肥大指数等。应用荧光实时定量RT-PCR和免疫组织化学的方法检测肾皮质TRAIL及其受体DR4、DcR2和NF-KB的mRNA和蛋白的表达情况。结果各时间点DM组大鼠尿蛋白量（24h）和肥大指数（肾质量，体质量）均显著高于NC组（P〈0．05）；血白蛋白在第8周末开始显著低于NC组（P〈0．01）；Scr、BUN于第16周末显著高于NC组（P〈0．01）。DM组大鼠TRAIL及其受体DR4mRNA在第4、8及12周末时表达均显著低于NC组（P〈0．01）；第16周末时表达显著高于NC组（P〈0．01）。DM组大鼠DcR2mRNA在第4、8及12周末时表达均显著高于NC组（P〈0．01）；NF-KB的基因表达均显著高于NC组（P〈0．05）。免疫组化结果显示TRAIL及其受体DR4、DcR2主要在肾小管表达，而在肾小球和脉管系统未见表达；NF-KB在肾小球和肾小管均有表达；TRAIL及其受体和NF-KB在各组表达趋势与PCR结果一致。结论TRAIL系统作为调节细胞凋亡的一组重要因子，参与了糖尿病肾病的发生发展。     

氯沙坦钾对糖尿病肾病大鼠肾组织p-JAK2、p-STAT3及VEGF表达的影响

目的：探讨氯沙坦钾对糖尿病肾病（diabetic nephropathy,DN）大鼠肾组织 p - JAK2、p - STAT3及 VEGF 表达的影响。方法：健康雄性 SD 大鼠30只随机分为正常对照组（C 组）、DN 模型组（DN 组）、氯沙坦钾组（L 组）。采用单次腹腔内注射链脲佐菌素（streptozotocin,STZ）法建立 DN 大鼠模型,周期12周。实验12周末检测大鼠血糖、Scr、BUN、24 h 尿蛋白定量;光镜及电镜下观察大鼠肾组织病理改变;采用免疫组化方法检测大鼠肾组织 p - JAK2、p - STAT3、VEGF 表达。结果：实验12周末,模型组大鼠血糖、Scr、BUN、24 h 尿蛋白定量均高于对照组（P ﹤0．05）,肾组织中 p - JAK2、p - STAT3、VEGF 表达均高于对照组（P ﹤0．05）;氯沙坦钾组大鼠 Scr、BUN、24 h 尿蛋白定量均低于模型组（P ﹤0．05）,肾组织 p - JAK2、p -STAT3、VEGF 表达均低于模型组（P ﹤0．05）。结论：氯沙坦钾可能部分通过调控 p - JAK2、p - STAT3及 VEGF 的表达而发挥肾脏保护作用。     

肾炎康复片对糖尿病肾病大鼠肾组织Podocalyxin表达的影响

目的：观察肾炎康复片对糖尿病肾病大鼠肾组织Podocalyxin表达、生化指标及病理改变的影响。方法：40只雌性Wistar大鼠随机分为正常对照组、DN模型组（模型组）、肾炎康复片治疗组（中药组）、氯沙坦钾治疗组（西药组）。复制糖尿病肾病大鼠模型,并予药物干预。分别观察不同时期各组大鼠血糖、24h尿蛋白定量,12周末大鼠肾组织Podocalyxin表达水平、Scr、BuN及肾脏组织病理变化。结果：12周模型组、中药组、西药组大鼠Podocalyxin蛋白表达均显著低于同期正常对照组（P〈0．05）,肾组织病理损伤明显,24h尿蛋白定量较正常对照组显著增高（P〈0．05）。12周末用药组大鼠肾组织Podocalyxin蛋白表达显著高于同期模型组（P〈0．05）,肾组织病理损伤较模型组明显减轻。血糖水平略低于同期模型组,但差异无统计学意义（P〉0．05）,24h尿蛋白定量、BUN、Scr水平显著低于同期模型组（P〈0．05）。结论：肾炎康复片能够上调糖尿病肾病大鼠肾组织Podocalyxin表达水平,对糖尿病肾病肾脏损伤有保护性作用。     

The protective effect of hydroxytyrosol on contrast - induced nephropathy and endoplasmic reticulum stress

Objective To investigate the expression of glucose-regulated protein 78(GRP78) and cysteine aspartic acid protease 12(Caspase - 12) and evaluate the endoplasmic reticulum stress (ERS) in rats with contrast - induced nephropathy (CIN), and observe the protective effects of hydroxytyrosol on CIN rats. Methods Eighty-four Wistar rats, (220±20) g, were randomly divided into control group, CIN group, hydroxytyrosol treated group (group C+H). At 12th, 24th, 48th, 72th day after the rats model were established, BUN and Scr were detected. ELISA were used to detect the expression of methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). HE staining were used to evaluate the pathological change of kidney. TUNEL were used to detect the apoptosis of tubular cells. Real-time PCR were used to detect the expression of GRP78 mRNA and Caspase-12 mRNA in tubular cells. Immunohistochemistry and Western blotting were used to detect the expression of GRP78 and Caspase - 12 protein in tubular cells. Results BUN, Scr, the mRNA and protein expression of GRP78, Caspase-12 in hydroxytyrosol treated group were higher than that in control group(P＜0.05), but were significantly lower than that in CIN group (P＜0.05). Pathological changes and the apoptosis of tubular cells in CIN group were more serious than that in hydroxytyrosol treated group (P＜0.05). Conclusions Endoplasmic reticulum stress may be associated with contrast-induced nephropathy. Hydroxytyrosol can protect kidney from contrast medium via reducing the endoplasmic reticulum stress.     

普罗布考防治大鼠顺铂急性肾损伤的实验研究

目的：复制大鼠顺铂急性肾损伤模型,研究氧化应激与核转录因子Sp1及凋亡的关系;探讨普罗布考对顺铂急性肾损伤的保护作用及作用机制。方法：24只SD雄性大鼠被随机分为生理盐水对照组、顺铂模型组、普罗布考干预组、普罗布考对照组,每组6只;检测尿N-乙酰-β-D-氨基葡萄糖甘酶（NAG）、血清肌酐（Scr）、血尿素氮（BUN）、肾组织匀浆液丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-Px）,光镜观察肾脏病理改变;采用免疫组化染色检测肾组织Sp1蛋白表达;采用TUNEL染色检测肾小管上皮细胞凋亡。结果：与生理盐水对照组和普罗布考对照组相比,顺铂模型组大鼠血清BUN和Scr,尿NAG酶,肾脏组织匀浆MDA含量显著升高,肾组织匀浆GSH-Px活力显著下降（P〈0.01）;肾脏指数、肾小管损伤分数和肾小管上皮细胞凋亡百分比均明显增加（P〈0.01）;肾组织Sp1蛋白的表达上调。采用普罗布考干预后血清BUN和Scr,尿NAG酶,肾脏组织匀浆MDA含量显著下降（P〈0.05）;肾组织匀浆GSH-Px活力显著升高;肾脏指数、肾小管损伤分数和肾小管上皮细胞凋亡百分比均明显降低（P〈0.01）;肾组织Sp1蛋白的表达下调。结论：氧化应激和核转录因子Sp1在顺铂所致大鼠肾毒性中起一定作用;普罗布考对顺铂所致大鼠肾毒性有保护作用,其机制可能与抗氧化、抑制肾小管上皮细胞凋亡、下调肾组织Sp1蛋白表达有关。     

红细胞生成素对慢性肾衰竭大鼠残肾组织归巢因子表达的影响

目的 观察红细胞生成素(EPO)对慢性肾衰竭(CRF)大鼠肾组织归巢因子表达的影响.方法 采用分阶段5/6肾切除制备大鼠CRF模型.实验动物随机分为3组:假手术组、CRF模型组和EPO治疗组.从第3周开始,治疗组大鼠每次皮下注射重组人EPO 50 IU/kg,每周3次,共6周.8周后检测各组大鼠血肌酐(Scr)、血尿素氮(BUN)、尿蛋白、血红蛋白(Hb)；采用实时荧光定量PCR、Western印迹和免疫组化方法检测残肾组织EPO及其受体(EPOR)、归巢因子及其受体(SDF-1、CXCR4、Ang-1、Tie2、SCF、c-Kit)的表达.结果 与模型组比较,EPO治疗可上调残肾组织归巢因子及其受体(SDF-1、CXCR4、Ang-1、Tie2、SCF、c-Kit)mRNA和蛋白的表达(均P＜0.05)；同时,EPO治疗还可上调残肾组织EPO及EPOR的mRNA和蛋白的表达(均P＜ 0.05).此外,EPO治疗还能下调大鼠Scr、BUN和尿蛋白水平(均P＜0.05),上调Hb水平(P＜0.05).结论 EPO能改善慢性肾衰竭大鼠的肾功能,这种作用可能与其激活残肾组织归巢因子而参与损伤肾脏的修复有关.     

Effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 on the expression of aquaporin 2 in kidneys of uremic rats

Objective To investigate the effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 [1,25(OH)2D3] on rat kidney aquaporin (AQP) 2 expression in 5/6 nephrectomized rats. Methods Twelve Sprague-Dawley rats underwent 5/6 nephrectomy surgery were divided into model group (n＝6) and 1,25(OH)2D3 group (n＝6) randomly; sham-operated rats only received the renal capsule stripping (control group, n＝6). Rats in 1,25(OH)2D3 group received 1,25(OH)2D3 (3 ng•100 g-1•d-1, ip) for 24 weeks. Serum and 24-hour urine specimens were collected for measurement of serum creatinine, arginine vasopressin (AVP) and urine protein. Animals were sacrificed at week 24 and kidneys were removed for routine pathological, immunohistochemistry and immunoblotting analysis. Results At week 24, plasma AVP level in 1,25(OH)2D3 and model group was much higher than that in control group (all P＜0.05), with no significant differences between the former two groups (P＞0.05). Lower serum creatinine and urinary protein were presented in 1,25(OH)2D3 group compared with the model group rats at week 24 (P＜0.05). Renal medullar fibrosis and inflammatory cell infiltration were improved significantly in 1,25(OH)2D3 group compared with model group (P＜0.01, P＜0.05). Immunohistochemistry analysis revealed abundant AQP2 and p-AQP2 expressed in the renal medulla of sham group, mainly in apical membrane of collecting duct cells. AQP2 expression in model group was down-regulated (P＜0.05) and p-AQP2 expression in apical membrane was reduced. AQP2 expression in 1,25(OH)2D3 group increased compared with model group, with increased p-AQP2 expression in apical membrane. Western blotting revealed same results of these expressions (all P＜0.05). Correlation analysis showed a negative correlation of AQP2 expression with urine volume, medullary fibrosis, and inflammatory cell infiltration (P＜0.05). Conclusion Long-term low-dose 1,25(OH)2D3 improves AQP2 expression and response to AVP in collecting duct, which may involve in the anti-polyuric effect of 1,25(OH)2D3 in uremic rat.     

Rg1、Rb1对糖尿病肾病大鼠肾脏保护作用及其对肾组织MCP-1 mRNA与蛋白表达的影响

目的：探讨人参皂苷Rg1、Rb1对糖尿病肾病大鼠肾组织MCP-1 mRNA及蛋白表达的影响。方法：75只雄性SD大鼠,正常组10只,其余采用腹腔内一次性注射STZ55mg/kg复制糖尿病大鼠模型,造模后血糖≥16.7mmol/L,尿糖（＋＋＋＋）以上者列入实验对象。随机分为模型组、厄贝沙坦组、Rg1大剂量组、Rg1小剂量组及Rb1组,各组分别给予相应药物灌胃治疗;每周测体重,于第4、8、12周末分别留尿用考马斯亮蓝法测24h尿蛋白。12周后处死大鼠,取血清检测BUN、Scr、TG。取肾脏称重,计算肾重体重比,将肾组织固定、包埋、切片后进行HE、PAM、Mallory和Masson染色,观察大鼠肾脏病理学变化。用免疫组化、原位杂交方法分别检测肾组织MCP-1 mRNA及其蛋白的表达。结果：各治疗组大鼠体重各周与模型组比较均有所增加,但无统计学差异。与模型组比,肾重/体重,Rb1组有统计学差异（P〈0.01）,Rg1大、小剂量组有统计学差异（P〈0.05）。24h尿蛋白定量与模型组比较,4周、8周,Rg1大、小剂量组及Rb1组有统计学差异（P〈0.01）;12周,Rg1大、小剂量组及Rb1组有统计学差异（P〈0.05）。Rg1、Rb1可以见降低BUN、Scr与TG,与模型组比较,BUN均有统计学差异（P〈0.01）,Scr为Rg1大剂量组及Rb1组有统计学差异（P〈0.01）,Rg1小剂量组（P〈0.05）;TG治疗组均有降低趋势,但无统计学差异。Rg1、Rb1均可以减轻DN大鼠肾脏病理损害。同时Rg1、Rb1还可以减少肾组织MCP-1蛋白及其mRNA的表达,与模型组比较,Rg1大、小剂量组及Rb1组肾组织MCP-1蛋白的表达均显著减少,具有统计学意义（P〈0.01）,Rg1大、小剂量组及Rb1组肾组织MCP-1 mRNA表达明显减少,其中Rg1大剂量组与模型组相比有统计学意义（P〈0.05）。结论：Rg1、Rb1可以改善DN大鼠肾脏功能、减轻肾脏病理损害,其具体机制可能与下调大鼠肾组织MCP-1 mRNA及蛋白表达水平有关。     

Effect of mycophenolate mofetil on CD26 expression in the kidneys of diabetic rats

Objective To investigate the expression of CD26 (dipeptidyl peptidase 4) in the kidney tissues of diabetic rats and the effects of mycophenolate mofetil (MMF) on the renal CD26 expression. Methods Wistar rats were randomly divided into three groups: normal control group (NC group, n=7), diabetic model group (DM group, n=7) and MMF-treated group (MMF group, n=7). Wistar rats were fed with high-sucrose-high-fat diet and injected with streptozotocin into abdominal cavity to induce diabetes. Sixteen weeks later, blood glucose (BG), blood urea nitrogen (BUN), serum creatinine (Scr), renal hypertrophy index (kidney weight/body weight) and 24 hour urinary protein (24Upro) were measured. The number of CD3+/CD4+ T cells in renal tissues were measured through flow cytometry. The expression of CD26 in kidney was examined by using Western blotting and immunohistochemistry. Results Compared with NC group, BG, BUN, Scr, kidney weight/body weight, 24Upro were significantly increased in DM group (P﹤0.05). Except BG and kidney weight/body weight, the above-mentioned parameters were lower in MMF group compared with that in DM group (P﹤0.05). Intrarenal CD3+/CD4+ T cells were significantly up-regulated in DM group compared with that in NC group (P﹤0.01). CD26 in renal tissue was mainly expressed in T lymphocytes of renal interstitium. CD26 expression in DM group was significantly higher than that in NC group, and also higher than that in MMF group (P﹤0.05). In DM group, CD26+ T lymphocytes infiltration of renal interstitium was positively correlated with 24Upro (r2=0.770, P﹤0.05). Conclusions CD26 is related with diabetic nephropathy. MMF maybe inhibit T lymphocytes infiltration to reduce the expression of CD26 in renal interstitium, thus protecting the kidney function.     

金雀异黄素对糖尿病大鼠肾皮质SMemb表达及细胞外基质的作用

目的探讨金雀异黄素(Gen,5,7,4’-三羟异黄酮,又称染料木黄酮)对糖尿病 (DM)大鼠肾组织系膜细胞(MC)表型和细胞外基质的作用。方法雄性SD大鼠45只,分为(1) 正常对照组:10只,普通鼠饲料自由饮食;(2)DM组:再分为4周组9只,8周组8只,普通鼠饲料自由饮食;(3)DM+Gen灌胃组:同样再分为4周组10只,8周组8只,普通鼠饲料自由饮食及 Gen 30 mg．kg-1·d-1。于实验4周、8周末禁食12 h,检测空腹血糖(FBG)、BUN、Scr,肾组织非肌肉型肌球蛋白重链(SMemb)mRNA表达、纤连蛋白(Fn)／肾重及基质金属蛋白酶(MMP)-2／组织基质金属蛋白酶抑制剂(TIMP)-I沉积结果。结果 DM大鼠FBG、BUN、Scr、Fn／肾重均高于正常对照组,以8周时最为显著(P<0．01);MMP-2／TIMP-1免疫组化半定量比值显著降低(P< O．01);SMemb mRNA／GAPDH mRNA半定量比值高于正常对照组,于4周达高峰,8周时下降 (0．794±0．037比0．708±0．029)。Gen干预后与同期DM组相比,肾功能部分恢复,SMemb mRNA／ GAPDH mRNA比值明显缩小(P<0．01);Fn／肾重也显著低于DM组(P<0．01);MMP-2／TIMP-1 比值与同期DM组相比也有显著增加(分别P<0．01或<0．05)。结论金雀异黄素可以减轻链脲佐菌素所诱导DM大鼠的肾皮质SMemb的表达,提高MMP-2／TIMP-1比值,改善糖尿病模型大鼠肾功能,可能有延缓糖尿病肾病(DN)进展的作用。     

活性维生素D通过调节糖尿病肾病大鼠巨噬细胞M1及M2表型活化防治足细胞损伤

目的 探讨活性维生素D（VD）能否通过调节肾组织巨噬细胞M1 及M2 表型活化从而防治糖尿病肾病（DN）大鼠足细胞损伤。 方法 利用腹腔注射链脲菌素（STZ）建立糖尿病大鼠模型。将SD 雄性大鼠按随机数字表法分为4 组：对照组1（NC-1 组,n=8）、对照组2（NC-2 组,n=8,对照+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）、DN 组（n=24）、DN+VD 干预组（VD 组,n=24,DN+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）,定期检测血糖、体质量,收集尿标本,分别于干预后8周、14周、18周末处死动物,检测Scr、BUN和尿蛋白变化;PAS染色观察肾脏病理改变;免疫组化法检测肾组织CD68+巨噬细胞浸润数量;Western 印迹法检测nephrin、podocin、CD68 以及M1巨噬细胞特异性标志物诱导型氮氧化物合酶（iNOS）、肿瘤坏死因子α（TNF-α）和M2巨噬细胞特异性标志物CD163、精氨酸酶1（Arg-1）、甘露糖受体（MR）表达。 结果 （1）与两对照组相比,DN 组Scr、BUN、24 h 尿蛋白量及肾小球系膜基质增生程度显著增加（P＜0.05）,podocin、nephrin蛋白表达下降（P＜0.05）。VD干预后能明显改善上述病理现象（均P＜0.05）。（2）与两对照组相比,DN组肾组织CD68⁺巨噬细胞浸润数量明显增加,呈时间依赖性。VD干预后能显著减少CD68⁺巨噬细胞浸润数量（P＜0.05）。（3）进一步确定肾组织巨噬细胞M1、M2活化表型发现,8周、14周、18周末DN组iNOS、TNF-α蛋白表达较对照组显著升高（均P＜0.05）,VD干预后能明显抑制同期DN肾组织iNOS、TNF-α表达（均P＜0.05）;8周、14周末VD组CD163、Arg-1、MR蛋白表达与DN组相比差异无统计学意义（均P＞0.05）,而18周末VD组CD163、Arg-1、MR蛋白表达较DN 组显著升高（均P＜0.05）,CD163/CD68 蛋白表达比例亦显著增加（P＜0.05）。（4）相关分析显示,M1 标志物iNOS 与nephrin、podocin 蛋白表达均呈负相关（r＝-0.707,P＜0.01;r＝-0.712,P＜0.01）;M2标志物CD163与nephrin、podocin蛋白表达均呈正相关（r＝0.627,P＜0.01;r＝0.613,P＜0.01）。 结论 活性维生素D具有调节巨噬细胞表型活化的能力,通过抑制巨噬细胞M1型活化并增强M2型活化,进而发挥足细胞保护作用。