大鼠缺血性肾损伤的细胞聚合糖性死亡与外源性凋亡

目的探讨大鼠肾缺血再灌注所致急性肾损伤时细胞聚合糖性死亡与外源性凋亡。方法应用免疫印迹技术、免疫组织化学染色技术以及光学和电子显微镜技术对缺血60min再灌注24h的大鼠肾组织进行观察和分析。结果免疫印迹分析结果表明,与sham组比较肾缺血再灌注后(AKI组)肾组织PARP-1、caspase-3和TNFRα表达增强。PARP-1、caspase-3免疫组化染色阳性细胞出现在缺血再灌注损伤肾组织,主要分布于肾小管,皮质和髓质外带的肾小管出现了大面积细胞坏死,表现为细胞肿胀,空泡形成,崩解脱落。在髓质外带肾小管坏死细胞之间存在着较多的凋亡细胞,细胞皱缩,核固缩。电镜下坏死细胞肿胀,细胞器也肿胀,崩解消失。凋亡细胞皱缩,界限清楚,核染色质固缩边聚。结论大鼠肾缺血60 min再灌注24 h部分肾小管上皮细胞发生聚合糖性死亡和外源性凋亡。     

大鼠缺血性急性肾损伤对肺脏超微结构的影响

目的　观察大鼠缺血性急性肾损伤对肺脏细胞超微结构的影响。 方法　应用光学显微镜和透射电子显微镜技术以及立体计量学方法观察急性肾缺血60 min再灌注24 h后的肺脏。 结果　部分肺泡I型上皮细胞发生了肿胀性坏死,II型上皮细胞也出现了肿胀性坏死,细胞内大量空泡出现。嗜锇性板层小体消失,肺泡内皮细胞出现了凋亡的表现。肺内组织出现了中性粒细胞浸润。 结论　大鼠肾脏急性肾缺血损伤可引起肺细胞坏死与凋亡,其中坏死损伤为多见。此外,炎性细胞浸润也是不可忽略的症候。     

缺血再灌流损伤对肾毛细血管内皮细胞的影响──细胞化学观察

应用常规电镜及过氧化氢细胞化学技术，观察了缺血再灌流对鼠肾毛细血管内皮细胞的损伤情况。缺血６０ｍｉｎ，可致毛细血管内皮细胞明显肿胀，过氧化氢细胞化学表现为在内皮细胞表面有少量电子致密物沉积。缺血６０ｍｉｎ复流１０ｍｉｎ，毛细血管内皮细胞表面有大量电子致密物沉积。缺血６０ｍｉｎ复流３０ｍｉｎ及６０ｍｉｎ，内皮细胞损伤加重，毛细血管内皮细胞与基底层之间裂开、翻起，甚至剥脱。毛细血管内皮细胞与肾小管上皮细胞之间有电子致密物沉积，甚至肾小管上皮细胞基底部也有致密物沉积。     

缺血再灌流损伤对肾毛细血管内皮细胞的影响——细胞化 …

应用常规电镜及过氧化氢细胞化学技术，观察了缺血再灌流对鼠肾毛细血管内皮细胞损伤情况后，缺血６０ｍｉｎ可致毛细血管内皮细胞明显肿胀，过氧化氢细胞化学表现为在内皮细胞表面有少量电子致密的沉积缺血６０ｍｉｎ复流１０ｍｉｎ毛细血管内皮细胞表面有大量电子致密物沉积，缺血６０ｍｉｎ复流３０ｍｉｎ及６０ｍｉｎ内皮细胞损伤加重，毛细血管内皮细胞与基底层之间裂开，翻起，甚至剥脱。毛细血管内皮细胞与肾小管上皮细胞之间     

大鼠急性肾损伤模型的建立及意义

目的探讨建立大鼠缺血再灌注急性肾损伤模型。方法采用切除大鼠右肾,左肾蒂夹闭45分钟,再灌注2、6、12、24 h测定血尿素氮(BUN)、肌酐(Scr)的水平,观察肾组织病理形态学改变,TUNEL法检测肾小管凋亡细胞,确定大鼠缺血再灌注急性肾损伤模型。结果 IRI组BUN、Scr在再灌注后2h开始上升,在24 h达到高峰;肾组织24 h后肾小管上皮细胞凋亡、坏死脱落,小管可见细胞碎片及管型,小管排列稀疏、紊乱,管腔明显扩张,肾小球无明显改变;与Sham组相比,IRI各亚组肾小管凋亡细胞指数与损伤评分,差异有统计学意义(P0.05),且随缺血再灌注时间延长及肾功能损伤的加重,肾小管凋亡细胞指数与损伤评分增加。结论切除大鼠右肾、夹闭左肾动脉的方法可成功建立缺血再灌注急性肾损伤动物模型。     

骨髓单个核细胞移植对肾缺血再灌注后肾小管坏死及细胞凋亡的影响

目的：观察和分析外源性骨髓单个核细胞（BMMNCs）对肾缺血再灌注诱发的肾小管坏死及凋亡的影响。方法：密度梯度离心法分离获取BMMNCs并行DAPI标记。制作SD大鼠肾缺血再灌注损伤模型，通过下腔静脉进行BMMNCs移植。分别于缺血再灌注后不同时相获取肾脏标本，荧光显微镜下观察DAPI的标记情况，HE染色做肾组织学检测，TUNEL法检测肾组织凋亡细胞，免疫组织化学染色观察增殖细胞核抗原（PCNA）的表达。结果：BMMNCs移植组大鼠肾脏组织中可见DAPI荧光细胞，部分存在于肾小管上皮组织中，未见明显细胞坏死及变性征象，其凋亡细胞计数显著减少，而PCNA阳性细胞数增多。结论：外源性BMMNCs移植可减少缺血再灌注诱发的肾小管上皮细胞的变性、坏死和凋亡，促进缺血再灌注损伤后肾小管上皮细胞的增殖，从而提示BMMNCs移植有助于缺血再灌注损伤后肾小管的修复及其结构完整性的维持。     

大鼠缺血再灌流后肾小管细胞凋亡与增殖的研究

目的：研究缺血性肾小管损伤与修复过程中细胞的增殖与凋亡。方法：暂时夹闭在鼠左肾动静脉４５分钟制肾缺血再灌流动物模型。应用ＨＥ染色、ＴＵＮＥＬ法、ＰＣＮＡ免疫组化及透射电镜观察。结果：肾缺血再灌流６小时至１周出现肾小管上皮细胞凋亡。缺血４５分钟增殖指数（ＰＩ）显著降低。缺血再灌流１２至７２小时ＰＩ显著升高。细胞凋亡的高峰出现在缺血再灌流１２小时。而ＰＩ的峰值在２４小时。结论：缺血引起的细胞增殖水平下     

樟柳碱、东莨菪碱、山莨菪碱对急性肾衰大鼠肾微血管损伤的保护作用

目的 :研究急性肾衰时肾微血管的损伤及樟柳碱、东莨菪碱、山菪莨碱对这些损伤的影响。方法 :采用左肾动脉注射油酸的方法建立大鼠急性肾衰模型 ,采用电镜、光镜及TTC显色观察大鼠急性肾衰不同时期肾微血管超微结构、肾组织结构及TTC显色的改变。结果 :左肾动脉注射油酸后 1 0min ,肾小球毛细血管内皮细胞坏死 ;肾小球毛细血管不同程度充血肿胀 ,在皮质与髓质交界区 ,可见小血管内充满大量红细胞 ;TTC显色呈鲜红色。注射油酸后 6h和 2 4h上述损伤加重 ,TTC显然出现典型的不显色苍白区。三种莨菪药物不同程度减轻了急性肾衰时肾微血管损伤 ,改善了肾组织缺血 ,减少了肾小管上皮细胞坏死数。结论 :急性肾衰时很早即出现肾微血管的损伤 ,三种莨菪药对急性肾衰时肾微血管和组织结构的损伤均有保护作用。     

大鼠肾缺血与再灌时近曲小管上皮细胞内钙含量变化

本实验采用细胞化学方法结合EDX微区分析及生化测试方法，从细胞水平研究了大鼠肾近曲小管上皮细胞内钙含量在缺血-再灌流损伤过程中的改变情况，并探讨了其机理。结果表明：缺血1h，可致细胞内钙含量增高，质膜Ca2 -ATPase活性下降；膜通透性无明显变化。再灌流期，细胞内钙含量持续增高；质膜Ca2 －ATPase活性在再灌流早期（2～4h）基本恢复至正常，在再灌流晚期（12～24h）下降；再灌流期膜通透性逐渐增大。肾小管上皮细胞内钙含量增加在缺血期、再灌流早期及晚期某机制并不相同。     

抗黏附治疗对大鼠急性肾损伤中树突状细胞迁移聚集的影响

目的：探讨黏附分子P-选择素、ICAM-1及树突状细胞(DC)在大鼠肾缺血再灌注损伤中作用，以及抗P-选择素单抗的抗黏附抑制及其防治效果。方法：建立肾缺血再灌注损伤大鼠模型，随机分为P-选择素单抗治疗组(n=20)和非治疗组(n=20)。按不同再灌注时间(1，3，6和24h)再分为4组，另设假手术组(n=5)作为对照。采用免疫组化LSAB法和免疫双染与荧光图像分析法，分别观察大鼠肾组织中P-选择素、ICAM-1表达及CD1a^ CD80^ DC分布变化；同时观察丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性以及细胞凋亡等改变。结果：①缺血再灌注1h起，P-选择素即以肾小管上皮细胞为主于肾内广泛高表达，至24h仍维持一定量水平；ICAM-1于缺血再灌注6h起持续以肾血管为主表达上调和明显增强。②CD1a^ CD80^ DC自再灌注1h起以肾小管间质为主分布数量逐渐增多，至24h达峰，并与大鼠血尿素氮和肌酐水平密切相关。③此外缺血再灌注后，大鼠肾组织MDA含量增加，SOD活性下降，出现明显的肾组织细胞凋亡。④经抗P-选择素单抗处理后，大鼠肾组织P-选择素和ICAM-1表达受到抑制，CD1a^ CD80^ DC分布及数量减少，MDA含量降低和SOD活性上升，细胞凋亡减少，以及肾组织病理损伤和肾功能也相应减轻和改善。结论：本研究提示，黏附分子介导树突状细胞迁移聚集参与了肾缺血再灌注病理损伤过程，而抗P-选择素单抗对此具有抗黏附调抑及防治效果。     

Topography of focal proximal tubular necrosis after ischemia with reflow in the rat kidney.

The topography of renal injury after ischemia-reflow was studied in intact rats by clamping the right renal artery for variable periods of time and examining the histologic appearance of the kidney in large 1-mu plastic sections. As reported by others, the straight portion of the proximal tubule (S3) was especially susceptible to ischemia-reflow injury. The pattern of focal necrosis produced in S3 by short (15-30-minute) and longer (45-60-minute) periods of arterial occlusion appeared related to gradients to oxygenation during reflow, in that more extensive injury was seen in areas remote from blood vessels, while perivascular tubules were protected. A similar pattern was seen in S1 and S2 after a longer period (45-60-minutes) of ischemia, which produced extensive but incomplete necrosis in these convoluted segments, with protected tubules lying within zones surrounding arcuate and interlobular arteries. The immediate postglomerular portion of S1, a tubular segment normally well supplied with oxygen, was an exception to this rule. Selective necrosis limited to this portion of the nephron appeared after only 15-30 minutes of ischemia, recalling the special sensitivity of S1 cells to inhibition of mitochondrial respiration. These results suggest that in different segments of the proximal tubule, injury after ischemia-reflow is determined not only by changes that occur during complete ischemia but also by the adequacy of oxygenation during the reflow period.     

丝裂素活化蛋白激酶亚类在急性缺血/再灌注肾损伤修复过程中的变化

目的:研究急性缺血/再灌注肾损伤修复时丝裂素活化蛋白激酶(MAPKs)亚类细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)的变化,探讨其在肾损伤修复中的意义。方法:夹闭大鼠双侧肾蒂造成急性缺血/再灌注肾损伤模型,应用电镜观察肾组织形态学改变,免疫组化法检测肾组织增殖细胞核抗原(PCNA)表达,采用特异性底物磷酸化结合免疫沉淀法测定肾组织的ERK、JNK活性。结果:急性缺血后再灌注2h肾小管上皮细胞腔面微绒毛重新出现,肾小管PCNA阳性细胞开始增加;缺血45min使ERK活性降低,再灌注5minERK活性完全恢复;缺血45min对JNK活性无影响,再灌注5minJNK活性增加并持续到再灌注2h。结论:MAPKs活性变化,特别是JNK活性增加参与了急性缺血/再灌注肾损伤早期细胞修复过程。     

TNF-α对肾脏近端小管上皮细胞caspase-3的调控作用及其机制

目的:探讨肿瘤坏死因子-α(TNF-α)对大鼠原代肾小管上皮细胞半胱氨酸天冬氨酸蛋白酶-3(caspase-3)激活与表达的调控以及核因子-κB(NF-κB)在其中的作用。方法:体外分离与培养大鼠肾小管上皮细胞,以TNF-α(10μg/L)按不同时间给予刺激,Western blotting检测caspase-3及其活化裂解片段cleaved caspase-3,以及I-κBα和磷酸化I-κBα(pI-κBα)蛋白水平。分别以TNF-α(10μg/L)处理24h、NF-κB特异性抑制剂Bay11-7082(5μmol/L)处理25h、Bay11-7082(5μmol/L)预处理1h后加入TNF-α(10μg/L)刺激24h,检测caspase-3及cleaved caspase-3的变化。结果:TNF-α刺激6至24h,cleaved caspase-3与caspase-3的相对比值显著高于对照组;而caspase-3蛋白水平在TNF-α刺激后6h无明显增高,12h低于对照组,24h回升;Bay11-7082处理组和Bay11-7082+TNF-α处理组的caspase-3蛋白表达与活化水平显著低于TNF-α处理组和对照组。结论:TNF-α促进大鼠原代肾小管上皮细胞caspase-3的激活与表达,这一过程与NF-κB的激活有关。     

Metabolic studies of postischemic acute renal failure in the rat

Postischemic acute renal failure was induced by 1 hr of clamping of the renal vasculature. Adenine nucleotide (ATP, ADP, AMP) and lactate (Lac) levels were measured after 0, 0.25, 1, 6, 24, and 48 hr of reflow to determine the time necessary for recovery to control levels. After 1 hr of ischemia with no reflow, [ATP] was 18% and [Lac] was 10-fold control levels. Control levels were restored after 24 hr of reflow. Variable ischemic times (5, 15, 30, 60, 90, and 120 min) followed by (1) no reflow or (2) 24 hr of reflow were also studied. [ATP] decreased to 25 and 13% of controls after 5 and 120 min of ischemia, respectively, and [Lac] increased to 5- and 13-fold controls after 5 and 120 min. Five to ninety minutes of ischemia followed by 24 hr of reflow resulted in a trend toward restoration of ATP and Lac levels; whereas, 120 min of ischemia followed by 24 hr of reflow resulted in death. The results indicate that: (1) In vivo ischemia results in a drastic and rapid shift in the ATP-ADP-AMP equilibrium; (2) the absolute concentration of ATP is not a reliable criterion of cell viability, but the ability to resynthesize ATP may be determinant in the reversibility of the lesion; (3) 1 hr of ischemia is reversible with respect to restoration of [ATP] and [Lac], but 24 hr of reflow are needed for restoration; and (4) ischemia for 90 min results in a metabolic derangement which is partially reversible in that metabolite levels are partially restored after 24 hr of reflow. However, 90 min of vascular clamping is not functionally reversible since the majority of animals exhibit severe azotemia and do not survive.     

Hexachloro-1:3-butadiene-induced renal tubular necrosis in the mouse

Administration of hexachloro-1:3-butadiene (HCBD) intraperitoneally at a dose of 50 mg/kg body weight caused necrosis of the proximal tubule of the mouse kidney. The earliest morphological changes detectable by light microscopy were observed after 4 h, and by 16 h extensive proximal tubular necrosis was seen throughout the cortex. Active tubular regeneration was apparent by day 5 and by day 14 substantial recovery of morphology had occurred. In the electron microscope mitochondrial swelling was seen 1 and 2 h after dosing in the S₁ and S₂ segments of the proximal tubule, whereas by 4 and 8 h the major pathological changes were confined to the lower S₂ and S₃ segments and consisted of mitochondrial swelling and cellular necrosis. The extent of renal injury and regeneration correlated well with measurement of renal function. In conclusion, HCBD produces necrosis of the S₂ and S₃ segments of the proximal tubule in the mouse, this pattern of injury is different from that seen in the rat where HCBD produces selective necrosis of the S₃ segment.     

The relationship between transforming growth factor β1 expression and cold ischemia injury of rat donor kidney

Objective

Cold ischemia injury represents an independent risk factor which favors chronic allograft nephropathy (CAN). In order to investigate the role of transforming growth factor-beta 1( (TGF-β1) in the progression of CAN, we studied the relationship between the expression of TGF-β1 and cold ischemia injury in the renal tubular epithelia of rat donor kidney.

Methods

A total of 24 Wistar rats were used in this study. In terms of the time of cryopreservation of donor kidney, the 24 Wistar rats were randomly divided into 3 groups: 0h group(control group), 24h group, 48h group. A block removal of donor kidney with in situ perfusion of cooling HC-A preservation solution was adopted. The rat kidney was preserved 0h, 24h and 48h at 0-4 °C respectively. The morphologic changes of proximal tubular epithelial cells in different cryopreservation time were observed under light microscope and transmission electron microscope. The expression of TGF-β1 mRNA and protein in proximal tubular epithelial cells of different cryopreservation time group were detected by in situ hybridization and immunohistochemistry analysis.

Results

1. In 24h group, part of the proximal tubular epithelial cells showed slight degeneration. In 48h group, the proximal tubular epithelial cells demonstrated severe hydropic degeneration and part of the cells developed necrosis and effluxion. 2. Only a small amount of TGF-β1 protein and mRNA were expressed in the renal tubular epithelial cells of 0h group. The positive unit (PU) value of TGF-β1 protein and mRNA were 6.37 ± 2.77 and 5.29 ± 2.15, respectively. As the cold ischemia time prolonged, the PU value of TGF-β1 protein increased at 24h group (10.20 ± 3.27) and 48h group (17.17 ± 3.96) . The PU value of TGF-β1 mRNA also increased at 24h group (11.31 ± 3.34) and 48h group (19.01 ± 3.53). There was the significant difference of TGF-β1 protein PU value or mRNA PU value among these groups(P < 0.05).

Conclusion

There was the significant correlation between the expression of TGF-β1 and the degree of cold ischemia injury. The results suggest that TGF-β1 might play the key role in regeneration and reparation of renal tubular epithelial cell injury. The overexpression of TGF-β1 might be one of the mechanisms that initiate chronic allograft nephropathy.     

己酮可可碱对抗兔肾脏缺血再灌注损伤的实验研究

目的: 研究己酮可可碱对抗兔肾脏缺血再灌注损伤作用。方法: 新西兰兔50只,随机分成5组:假手术组、缺血再灌注组(I/R)、I/R+己酮可可碱组、I/R+低温组、I/R+己酮可可碱+低温组。除假手术组外,其余4组均用动脉夹夹闭左侧肾动脉60min后恢复血流灌注,24h后取左肾分别检测肾组织匀浆的SOD、MDA和BUN、SCr水平的变化,并作病理观察。结果: I/R组肾小管出现水样变性、出血坏死,出血坏死组织中大量炎性细胞浸润,近曲小管上皮细胞线粒体高度肿胀,嵴极其紊乱、模糊、甚至消失;I/R+己酮可可碱组和I/R+低温组损伤减轻;I/R+己酮可可碱+低温组和假手术组形态正常。再灌注24h后I/R组BUN、SCr、MDA水平明显高于其它各组(P＜0.05),SOD活性明显低于其它各组(P＜0.05),I/R+低温组和I/R+己酮可可碱组BUN、SCr、MDA水平明显低于I/R组(P＜0.05),SOD活性明显高于I/R(P＜0.05),低温+己酮可可碱组的上述指标与假手术组无显著差异(P>0.05)。结论: 己酮可可碱有对抗兔肾脏缺血再灌注损伤的作用。