阿托伐他汀调控PERK/eIF2α/CHOP通路减轻造影剂诱导的大鼠肾小管上皮细胞凋亡

目的研究造影剂肾病大鼠肾脏中葡萄糖调节蛋白78(GRP78)、内质网调节激酶(PERK)、真核起始因子2α(eIF2α)及C/EBP同源蛋白质(CHOP)的表达情况,探讨内质网应激在造影剂肾病发病中的作用及阿托伐他汀的干预作用。 方法60只大鼠随机分为4组：对照组、模型组和高、低剂量阿托伐他汀组(80 mg,40 mg),每组15只。分别于注射造影剂后24、48、72 h留取血清;检测各组大鼠的血清尿素氮(BUN)、血清肌酐(Scr);TUNEL法及Western印迹法测casepase-3的表达检测肾小管上皮细胞凋亡;免疫组化和Western印迹法检测各组大鼠肾组织GRP78、p-eIF2α、p-PERK及CHOP的表达。 结果与对照组相比,模型组大鼠BUN、Scr显著升高,细胞凋亡严重,GRP78、p-eIF2α、p-PERK及CHOP的表达均显著升高(P< 0.05);与模型组相比,高、低剂量阿托伐他汀组,BUN、Scr显著下降,凋亡指数降低,GRP78、p-eIF2α、p-PERK及CHOP的表达显著下调,但仍高于对照组,差异均达到统计学意义(P<0.05);高、低剂量阿托伐他汀组之间上述各指标差异均不显著。 结论PERK/eIF2α/CHOP通路介导的内质网应激可能参与大鼠造影剂肾病的发生发展;阿托伐他汀在造影剂诱导的肾脏损伤中发挥保护作用,这可能与其调节PERK/eIF2α/CHOP通路,从而减轻内质网应激有关。     

制大黄-川芎药对对造影剂肾病大鼠肾小管上皮细胞凋亡的影响

目的：探讨制大黄-川芎药对对造影剂肾病（CIN）大鼠肾小管上皮细胞凋亡的影响及机制。方法：将32只雄性SD大鼠分为正常组（A组）、模型组（B组）、药对高剂量组（C组）、药对低剂量组（D组）。C、D组于造模前7天每日灌胃药对水煎液,灌胃量分别为成人标准体重（60kg）常规用量50、20倍。造模后24h处死动物,测定血清肌酐、尿素氮,HE染色观察肾脏病理改变,TUNEL染色检测肾小管上皮细胞凋亡,western印迹检测肾组织Caspase-3表达。结果：建模后24h与A组相比,B组血清肌酐、尿素氮均明显升高（P〈0.01）;病理形态学检测提示模型大鼠发生明显肾间质水肿、肾小管上皮细胞胞浆空泡样变、肾小管上皮细胞凋亡（P〈0.01）,Caspase-3蛋白表达显著增多（P〈0.01）。与B组相比,C、D组血清肌酐、尿素氮明显回落（P分别〈0.01or〈0.05）,肾脏病理改变显著为轻,肾小管上皮细胞凋亡指数显著减少（P〈0.01）,Caspase-3蛋白表达明显降低（P〈0.01）。结论：caspase3依赖的肾小管上皮细胞凋亡参与了大鼠造影剂肾病的发生,制大黄-川芎药对能通过抑制caspase3抑制肾小管上皮细胞凋亡,并进而保护CIN大鼠肾功能。     

STF-083010对肾脏缺血-再灌注损伤的保护作用

目的 观察STF083010对急性肾缺血-再灌注损伤的作用,探讨其损伤保护作用的机制.方法 选择健康雄性SD大鼠30只,随机分为假手术组(打开腹腔)、I-R组(建立大鼠肾I-R损伤模型)与STF083010组.分别在缺血-再灌注24h后处死大鼠,取血液和肾组织.全自动生化仪检测各组血清尿素氮(BUN)及肌酐(Scr)水平.PAS染色观察大鼠肾组织病理变化,免疫组化检测肾脏组织中XBP1、GRP78蛋白的表达.Quantitative real-time PCR(QPCR)测定大鼠肾组织标本中XBP1、GRP78 mRNA水平.结果 I-R组肌酐、尿素氮水平与假手术组相比差异均有统计学意义(P＜0.05).STF-083010组与I-R组相比,差异亦有统计学意义(P＜0.05).PAS病理图片可见STF-083010组肾小管损伤较I-R组明显减轻(P＜0.05);免疫组化检测显示STF-083010组XBP1的表达较I-R组明显降低(P＜0.05),STF-083010组GRP78蛋白的表达较I/R组明显升高;QPCR结果显示STF-083010组XBP1 mRNA水平较I-R组明显降低(P＜0.05),STF-083010组GRP78 mRNA较I-R组明显升高(P＜0.05).结论 STF-083010可以对大鼠肾脏缺血-再灌注损伤性保护作用.     

Effect of astaxanthin intervention on contrast-induced acute kidney injury in experimental rats

Objective To explore the protective effect and mechanism of astaxanthin (AST) on the acute kidney injury induced by iohexol in rats. Method Thirty rats were randomly divided into five groups: control group (Ctrl); iohexol group (CM); astaxanthin group (AST, 100 mg/kg), low astaxanthin dose group (LAST+CM, 50 mg/kg) and high astaxanthin dose group (HAST+CM, 100 mg/kg), 6 in each group. The rats in AST, LAST+CM, HAST+CM groups were administrated with AST by oral gavages using an intubation needle for 10 consecutive days. The rats in Ctrl and CM groups rats in Ctrl, CM groups were given with dissolvant instead in equal volume. Except for the Ctrl and AST groups, on day 8, rats were given indomethacin, L-NAME and iohexol in their femoral vein under chloral hydrate anesthesia to build a contrast induced-nephropathy (CIN) model. At the end of the experiment (72 h after CIN induction), all rats were sacrificed. The Scr level, BUN level, renal histology, renal tissue activities in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), Glutathione (GSH) and level of malondialdehyde ( MDA ) were performed. Apoptosis of renal cells was detected by Bcl-2, Bax and Caspase-3 p17 with Western blot. Results Compared with Ctrl group, the levels of Scr, BUN were significantly increased in CM group (all P＜0.01); while compared with CM group, the indicators were decreased in treatment groups (P＜0.01). Renal tubular structure damage, medulla congestion, loss of brush border, vacuolar degeneration, apoptosis and proteinaceous casts were observed in the CM group, and the renal injury scores were higher compared with Ctrl group (P＜0.05), however, administrated with AST could significantly improve the changes (P＜0.05). Oxidative stress indicators showed that MDA level were increased while SOD, GPx, GSH activities were significantly decreased at CM group (all P＜0.05), and the indicators above were ameliorated in treatment groups (all P＜0.05). Western blot showed that the expression of Bcl-2 was down-regulated while the Bax, Caspase 3 p17 was up-regulated respectively at CM group (P＜0.05), while the HAST+CM group could prevent the changes. Conclusions Iohexol can results in oxidative stress increased in kidney, which activate Caspase-3 p17 signal path, down-regulated Bcl-2 expression, up-regulated Bax expression respectively, and lead to cell apoptosis. AST can ameliorate the changes, especially with high AST dose, which suggest that the possible protection mechanism is by ameliorating oxidative stress and inhibiting apoptosis pathways.     

Lisinopril suppresses the endoplasmic reticulum stress associated tubular cell apoptosis induced by proteinuria in adriamycin nephropathy

Objective To investigate the role of endoplasmic reticulum stress in tubular epithelial cell apoptosis in chronic proteinuria rat model and the effect of lisinopril intervention. Methods Adriamycin nephropathy was induced in male Wistar rats (n=12) by a single injection of adriamycin at 2 mg/kg body weight. Rats were then randomly assigned to model group or treatment group, to which distilled water or lisinopril were administered respectively for 12 weeks. Six normal rats serving as controls were administered distilled water. 24 h urine samples were collected at week 4, 8, 12 and the urine protein was measured. At the end of study, serum was obtained and physiological parameters (serum creatinine, urea, total protein and albumin) were measured. Renal tubular epithelial cell apoptosis was assessed by TUNEL. GRP78, CHOP protein expression in kidney was quantified by immunohistochemistry and Western blotting. Results Compared to control group rats, increased proteinuria was observed in model group rats at week 4, 8, 12 (P＜0.05). Lisinopril treatment attenuated urine protein excretion significantly (P＜0.05). At week 12, hypoalbuminemia was detected in model group rats (P＜0.05), whereas the condition was alleviated by lisinopril (P＜0.05). There were no significant differences of serum creatinine, urea and total protein in each group (P＞0.05). Compared to control group rats, increased TUNEL positive tubular epithelial cells and tubular GRP78 and CHOP expression were also observed in model group rats (P＜0.05); however, these conditions in the kidney were significantly decreased in treatment group (P＜0.05). Conclusions Endoplasmic reticulum stress may be involved in the process of tubular epithelial cell apoptosis induced by proteinuria. Lisinopril may attenuate tubular epithelial cell apoptosis through regulating this signal pathway.     

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.     

Expression of 4-hydroxynonenal in the kidney of diabetic rats and the effect of probucol

Objective To investigate the expression of 4-hydroxynonenal (4-HNE) in the kidney of diabetic rats and the effect of probucol. Methods The rats were being intraperitoneal injected with STZ (60 mg/kg) to establish diabetic models. Then diabetic rats were randomly divided into diabetic group (group D, n＝24), probucol treated group (group P, n＝24). Normal rats were taken as control group (group C, n＝24). Rats in group P were treated by probucol (110 mg·kg-1·d-1); rats in group D and group C were given equal volume water instead. Scr, BUN, triglyceride (TG), total cholesterol (TC) and 24-hour urinary proteinin were measured at the 4th, 8th and 12th week. PAS staining and HE staining were used to evaluate the pathological changes of the kidney. The immunohistochemistry and Western blotting were used to detect the expression of 4-HNE in renal tissue. Results Levels of Scr, BUN, TG, TC and 24-hour urinary protein in group D were higher than those in group C at the 4th, 8th and 12th week(all P＜0.05); Levels of Scr, BUN, TG, TC and 24-huor urinary protein in group P were lower than those in group D at 4th, 8th and 12th week (all P＜0.05). The pathological changes of the kidney in group D were more serious than that in group P. The expression of 4-HNE in group Dwerehigher than group C at the 4th, 8th and 12th week (all P＜0.05); The expression of 4-HNE in the kidneys of group P decreased significantly compared to that of group D at the same time (P＜0.05). Conclusions As an indicator of lipid peroxidation, the expression of 4-HNE significantly increases in the kidney of diabetic rat. Probucol may protect the diabetic kidney through decreasing the expression of 4-HNE and the level of lipidperoxidation.     

替普瑞酮对大鼠肾脏缺血再灌注损伤的保护作用

目的 探讨替普瑞酮对肾脏缺血再灌注损伤的保护作用和可能机制。 方法 应用替普瑞酮(400 mg/kg)诱导雄性SD大鼠肾脏高表达热休克蛋白72（HSP72）。以钳夹大鼠左肾蒂45 min后，松开血管夹并切除右肾，建立大鼠缺血再灌注肾脏损伤模型。假手术组为打开腹腔，分离肾血管周围组织，但不钳夹血管。模型建立后24 h处死大鼠，留取血清测血肌酐（Scr）和尿素氮（BUN）。肾组织石蜡切片行PAS染色，以损伤肾小管所占百分比评分法评估肾组织肾小管损伤程度。TUNEL法检测缺血再灌注损伤时肾脏细胞凋亡的发生情况。Western印迹检测X连锁凋亡抑制蛋白（XIAP）的水平。 结果 缺血再灌注损伤可导致急性肾衰竭，表现为血Scr、BUN明显升高（P < 0.01）；PAS染色显示外髓部有大片肾小管坏死，甚至出现基底膜裸露；TUNEL染色中肾小管上皮细胞TUNEL阳性细胞数明显增多（P < 0.01）；Western印迹结果显示，肾组织XIAP蛋白水平明显降低（P < 0.01）。替普瑞酮处理后，肾组织HSP72表达水平明显增高（P < 0.01）；缺血再灌注所致的肾脏损伤明显改善，包括肾小管的损伤、细胞凋亡以及肾功能。此外，替普瑞酮可稳定肾组织XIAP的蛋白水平（P < 0.05）。 结论 替普瑞酮可诱导肾脏高表达HSP72。替普瑞酮可能通过减少肾脏XIAP蛋白的降解，抑制细胞凋亡，减轻缺血再灌注的肾脏损伤。     

Relationship of angiotensin II type 1 receptor autoantibody and renal cell apoptosis induced by caspase-12 in diabetic nephropathy rats

Objective To study the relationship of angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA) and renal cell apoptosis induced by caspase-12 in diabetic nephropathy (DN) rats. Methods High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (35 mg/kg) were utilized to establish DN rat model. Serum AT1-AA was detected by enzyme-linked immunosorbent assay (ELISA) and renal cell apoptosis was detected by TUNEL staining. Furthermore, the mRNA levels of the endoplasmic reticulum stress (ERS) chaperone protein glucose regulated protein 78 (GRP78) and ERS-associated apoptosis protein caspase-12 were measured by real-time quantitative PCR. Additionally, the levels of GRP78 and caspase-12 protein were measured by Western blotting. Results The renal cell apoptosis rate in DN group was increased significantly (P＜0.01), and the renal cells apoptosis rate in AT1-AA positive DN group was higher than that in AT1-AA negative DN group [(20.05±1.71)% vs (13.24±4.93)%, P＜0.01]. The mRNA expressions of GRP78 and caspase-12 in DN group, in comparison to NC group, were increased significantly (P＜0.01), as well as the proteins (P＜0.01). And the expression of these mRNA and proteins had significant increment in AT1-AA positive DN rats when compared with AT1-AA negative DN rats (P＜0.05). Conclusions AT1-AA can induce ERS in the renal tissue of DN rats, and promote renal cell apoptosis likely via the modulation of caspase-12 signaling pathway.     

川芎嗪与N-乙酰半胱氨酸抑制对比剂肾病大鼠肾小管上皮细胞凋亡的比较

目的评价川芎嗪抑制对比剂。肾病大鼠。肾小管上皮细胞凋亡的效应。方法建立对比剂肾病大鼠模型,分正常组（A）、模型组（B）、N-乙酰半胱氨酸组（C）、川芎嗪组（D）。C、D组在造模前3d每日分别腹腔注射150mg／kgN-乙酰半胱氨酸、80mg／kg川芎嗪。测定肾功能,观察。肾脏病理改变,TUNEL染色检测肾小管上皮细胞凋亡,Western印迹检测肾组织Caspase3表达。结果与A组相比,B组血肌酐、尿素氮、血清胱抑素C明显升高,肾间质水肿、肾小管上皮细胞空泡样变及细胞凋亡（P〈O．01）、Caspase-3蛋白表达增多（P〈0．05）。与B组相比,C、D组血肌酐、尿素氮、血清胱抑素C明显回落,病理改变显著减轻,肾小管上皮细胞凋亡指数显著减少（P〈0．01）,Caspase3蛋白表达降低（P〈O．05）,C、D组在上述检测中差异不显著（P〉0．05）。结论Caspase3依赖的肾小管上皮细胞凋亡参与了对比剂肾病的发生,川芎嗪通过抑制Caspase3抑制。肾小管上皮细胞凋亡并保护大鼠肾功能,效果与N_乙酰半胱氨酸组相近。     

内质网应激反应在严重烧伤大鼠心肌细胞凋亡中的作用

目的内质网应激反应(endoplasmic reticulum stress,ERS)介导的凋亡是真核细胞重要凋亡途径之一,通过观察严重烧伤大鼠心肌ERS不同通路蛋白表达变化,探讨其在心肌细胞凋亡中的可能作用。方法雄性7周龄Wistar大鼠64只,体重200～220 g;随机分为两组,每组32只。实验组大鼠背部制备30%体表面积Ⅲ度烫伤;对照组制备假伤模型。伤后1、4、7、14 d两组各处死8只大鼠取心肌组织,透射电镜观察心肌超微结构变化,TUNEL法检测心肌细胞凋亡,Western blot检测ERS相关蛋白,如葡萄糖调节蛋白78(glucose regulated protein 78,GRP 78)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、半胱氨酸天冬氨酸蛋白酶12(Caspase 12)剪切体表达变化。结果大鼠均存活至实验结束。透射电镜观察示实验组大鼠心肌细胞呈凋亡改变。伤后各时间点实验组心肌细胞凋亡指数均明显高于对照组(P<0.05),伤后1、4、7 d凋亡指数逐渐升高,14 d时下降,各时间点间比较差异均有统计学意义(P<0.05)。实验组心肌细胞GRP78、CHOP及Caspase 12剪切体蛋白表达持续升高,其中各时间点GRP 78及Caspase 12剪切体表达均较对照组显著升高,差异有统计学意义(P<0.05);除伤后1 d外,其余各时间点实验组CHOP蛋白表达均较对照组升高(P<0.05)。结论严重烧伤后大鼠心肌发生ERS,其中CHOP、Caspase 12介导的凋亡通路活化,ERS可能是心肌细胞凋亡的途径之一。     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

目的 探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。 方法 健康雄性Wistar大鼠25只,按随机数字表法分为UUO模型组（n=18）和假手术组（n=7）,UUO模型组行左侧输尿管结扎术,假手术组仅分离输尿管不结扎,分别于术后3 d、7 d、14 d处死各组大鼠,行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织羟脯氨酸（HYP）含量;免疫组化法检测α平滑肌肌动蛋白（α-SMA）;原位末端标记法（TUNEL）与DNA电泳观察肾小管间质细胞凋亡情况;RT-PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化;Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase-3）和GRP78的蛋白表达变化。 结果 与假手术组比较,UUO模型组肾脏病理改变加重,肾间质纤维化程度随梗阻时间延长逐渐加重,肾组织HYP含量显著升高（P < 0.05）,肾组织α-SMA也在肾小管间质细胞广泛表达,TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义（均P < 0.01）;在此后观察期间内GRP78 mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3 d即有显著上调（P < 0.05）,且随着梗阻时间延长进行性升高,于术后7 d、14 d增多更为显著,与假手术组差异均有统计学意义（P < 0.05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r = 0.657,P < 0.01;r = 0.714,P < 0.01）。 结论 UUO早期即可诱导ERS标志蛋白表达变化,触发ERS。长期ERS可诱导肾小管间质细胞凋亡;caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。     

Effect of intermedin on microvascular injury of unilateral ureteral obstruction rats

Objective To observe the effect of intermedin(IMD) on microvascular injury of renal fibrosis in unilateral ureteral obstruction (UUO) rat model. Methods Seventy-two male Wistar rats were randomly divided into two groups: the sham - operation group (n=24) underwent the left ureteral dissection, the other 48 rats were made as unilateral ureteral obstruction models and sub - divided into model group(UUO, n=24) and IMD group (n=24). At the 7, 14, 21, 28 day after the operation, 6 randomly - selected rats from each of the three groups respectively were blooded by abdominal arotic and their obstructive kidneys were taken out. The renal histopathological changes were observed through HE and Masson staining, the contents of BUN, Scr and cystatin C (CysC) of the obstructive kidneys were determined, the expressions of transforming growth factor - β1 (TGF - β1), α-SMA, bone morphogenetic protein-7 (BMP-7), E-cadherin, thrombospondin 1 (TSP-1) and vascular endothelial growth factor (VEGF) were detected by RT - PCR and immunohistochemistry. Results Compared with the sham-operated group, the pathological changes of kidney in the model group showed that the degree of fibrosis was obvious, tubular interstitial damage aggravated, the levels of BUN, Scr, CysC in the model group increased (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA, TSP-1 increased (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF decreased (P＜0.05). Compared with the UUO group, renal tubular damage, interstitial fibrosis in the IMD group were lighter, the levels of BUN, Scr, CysC in the IMD group were lower (P＜0.05), the mRNA expression and protein content of TGF-β1, α-SMA,TSP-1 were down-regulated (P＜0.05), while the levels of BMP-7, E-cadherin and VEGF were up-regulated (P＜0.05). Conclusion IMD can ameliorate the renal interstitial fibrosis, and the mechanism may be related to the fact that VEGF mediated by IMD can reduce vascular injury.     

AMPK attenuates inflammation to reduce fibrosis induced by acute ischemia reperfusion injury in mice

Objective To observe the effect of adenosine monophosphate activated protein kinase (AMPK) on attenuating inflammation in fibrosis induced by acute ischemia reperfusion injury (IRI) in mice. Methods Forty eight male C57BL/6 mice were randomly divided into four groups: sham operation group (sham group), IRI group, AMPK inhibitor+IRI group (AMPK/IRI group) and normal saline+IRI group (NS/IRI group), 12 mice each group. The mice with renal IRI were occluded for 30 min through clipping bilateral renal pedicle, then released renal perfusion. Mice in sham group were performed the separation of renal pedicle without clipping. Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI. At the 2 d after operation, 6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr. The renal histopathological changes were observed through HE staining. The mRNA expression of IL-1β, IL-6 and TNF-α was detected by real time PCR, and the level of AMPK phosphorylation was detected by Western blotting. At the 14 d after operation, Collagen 1 (COL1), α-SMA and fibronectin (FN) were detected by immunofluorescence and Western blotting in 6 remained mice from each group. The degree of kidney fibrosis was observed through sirus red staining. Results Compared with those in sham group, tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d after operation (all P＜0.05), and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group (all P＜0.05); the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d (all P＜0.05). Compared with those in IRI group, in AMPK/IRI group tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (all P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d (all P＜0.05), and the level of AMPK phosphorylation was decreased (P＜0.05). Moreover, the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d in AMPK/IRI group (all P＜0.05). Conclusions AMPK can ameliorate the acute renal ischemia reperfusion injury induce fibrosis in mice, and the mechanism may be related to the decrease of inflammatory reaction.     

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.     

外源性硫化氢在大鼠肾脏缺血再灌注损伤中的保护作用

目的 观察不同剂量外源性硫化氢(H2S)供体硫氢化钠对大鼠肾脏缺血再灌注损伤( IRI)的保护作用.方法 健康雄性Wistar大鼠28只随机分为4组,即假手术组( Sham)、肾缺血再灌注(IR)组、硫氢化钠(NaHS)高剂量组、硫氢化钠低剂量组.大鼠右肾切除后,以NaHS作为硫化氢的供体,NaHS高、低剂量组分别经左肾动脉插管,按照1.5 μmol/min、300 nmol/min的剂量连续15 min给药,假手术组及IR组给予同体积生理盐水.停药5 min 后,NaHS组和IR组用无损伤微动脉夹夹闭左侧肾蒂45 min后解除阻断,建立大鼠急性IRI模型,假手术组不夹闭左肾动脉,其他操作同模型组.于肾脏恢复血流24h时留取血和肾组织标本,检测血清尿素氮(BUN)、血肌酐(Scr);半定量分析肾脏病理损伤;检测肾组织H2S生成率;采用实时定量PCR法检测胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE )mRNA表达.结果 与假手术组相比,IR组H2S生成率显著降低(P＜0.01);CBS、CSE mRNA表达显著下降(P＜0.01 );Scr、BUN显著升高(P＜0.01);肾脏病理表现为急性肾小管坏死,且最严重.与IR组相比,NaHS预处理组H2S生成率升高(P＜0.05);CBS、CSE mRNA表达升高(P＜0.01 );Scr、BUN降低(P＜0.01);病理损伤明显减轻.NaHS两个剂量组之间差异无统计学意义.结论 外源性H2S对大鼠IRI具有保护作用.     

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.     

缬沙坦对糖尿病肾病大鼠肾间质纤维化的作用

目的 探讨缬沙坦在防治糖尿病肾病(DN)大鼠肾间质纤维化( RIF)中的作用及其机制.方法 54只大鼠随机被分为对照组(C组)、DN模型组(D组)和缬沙坦治疗组(T组).D组和T组大鼠分别给予链脲菌素( STZ)一次性腹腔注射建立糖尿病大鼠模型.造模后T组给予缬沙坦混悬液40 mg· kg-1·d-1,分别于实验第4周、第8周和第12周末测血糖、血浆白蛋白、Scr、尿蛋白.Masson染色观察RIF面积.免疫组化法检查肾组织缺氧诱导因子1α( HIF-1α)、金属蛋白酶1组织抑制剂(TIMP-1)、基质金属蛋白酶9(MMP-9)的表达.实时荧光定量PCR测定其mRNA表达.结果 与C组比较,D组及T组大鼠24 h尿蛋白量、Scr均显著升高,肾组织RIF面积、HIF-1α、TIMP-1蛋白及mRNA表达均显著增加,血清白蛋白及肾组织中MMP-9蛋白及其mRNA表达均明显减少,差异均有统计学意义(均P＜0.05).与同时间点D组比较,T组在实验第8周末、第12周末24h尿蛋白、Scr均显著降低,肾组织中RIF面积、HIF-1α、TIMP-1蛋白及其mRNA表达均显著减少,血清白蛋白及肾组织中MMP-9及其mRNA表达均显著增多,差异均有统计学意义(均P＜0.05).结论 缬沙坦可能通过下调HIF-1α、TIMP-1表达,上调MMP-9表达,延缓肾间质纤维化.     

羟苯磺酸钙对慢性马兜铃酸肾病大鼠肾组织CD34和血管性血友病因子表达的影响

目的 观察羟苯磺酸钙对慢性马兜铃酸肾病(CAAN)大鼠肾小管周毛细血管内皮细胞特异抗原CD34和血管性血友病因子(vWF)表达的影响,探讨羟苯磺酸钙改善CAAN大鼠模型肾脏微循环障碍的作用和机制.方法 关木通水煎剂灌胃12周制作马兜铃酸肾病大鼠模型,随机分成未治疗组和治疗组.未治疗组(n=8)予饮用水灌胃4周;治疗组(n=8)予羟苯磺酸钙灌胃4周.另设健康对照组(n=8).实验第16周处死所有大鼠,留取血、尿、肾组织标本行生化、病理及免疫组织化学检查.结果 与未治疗组比较,治疗组肾功能明显改善,肾间质纤维化程度减轻[(38.22±5.17)×103比(69.97±17.69)×103,P＜0.01],CD34阳性表达显著增加[(16.72±4.17)×103比(3.19±1.40)X103,P＜0.01];vWF阳性表达显著减少[(10.16±1.67)×103比(18.66±4.65)×103,P＜0.01)].结论 羟苯磺酸钙能增加CAAN大鼠肾组织CD34的阳性表达,增加肾小管周围毛细血管密度;减少CAAN大鼠肾组织vWF的阳性表达,减少微血栓形成.     

Expression of NLRP3 inflammasome in the BSA-overloaded rats kidney

Objective To observe NLRP3 inflammasome expression and inflammatory cells infiltration in the BSA-overloaded rats kidney, and to investigate the potential mechanism of renal injury induced by proteinuria. Methods After unilateral right nephrectomy, eighteen healthy male Wistar rats were randomly divided into two groups: protein overload nephropathy model group (n=10), treated with intraperitoneal injections of bovine serum albumin (BSA); control group (n=8), treated with intraperitoneal injections of 0.9% saline for 9 weeks. Body weigh were measured every week and 24 h urine were collected in 0, 2, 5, 7, 9 week. The plasma levels of blood total protein (TP), albumin (Alb), serum creatinine (Scr) and blood urea nitrogen (BUN) were determined by automatic analyzers. Renal pathological changes were evaluated by PAS and Masson stains. Immunohistochemical staining was used to detect the expression of NLRP3, caspase-1, IL-1β, and IL-18, as well as the types of inflammatory cells. The NLRP3, caspase-1, IL-1β, and IL-18 protein and mRNA levels were also analyzed by Western blot and real-time PCR in two groups. Results It was found that there was a significant increase of proteinuria and BUN in model group compare to that in control group (all P＜0.05). However, there were no significant changes in body weight, TP, Alb and Scr between the two groups. Morphological study demonstrated that renal tubular epithelial cell injury, proteinaceous casts in tubular lumen, accompanying with the dominant macrophages and lymphocytes infiltration in interstitium in model group. The immunohistochemistry showed that there were more T (CD3+), B cells (CD20+) and macrophages (CD68+) in renal interstitium in model group than that in control group (P＜0.05). Tubulointerstitial injury score was higher than that of the control group (P＜0.05). Immunohistochemistry, Western blot and real-time PCR all showed that the expression of NLRP3, caspase-1, IL-18 and IL-1 β were significantly increased compared to those in control group (P＜0.05). Furthermore, there were significant correlations between proteinuria and IL-1β/IL-18 expression (P＜0.05). Conclusion NLRP3 inflammasome activation is involved in tubulointerstitial inflammation caused by proteinuria.