肾炎益气液对大鼠肾毒血清性肾炎肾功能、肾组织病理学的影响

目的:观察肾炎益气液对肾毒血清性肾炎大鼠肾功能及肾组织学变化的影响。方法:应用大鼠肾毒血清性肾炎模型,以尿蛋白定量,血清肌酐、尿素氮测定反映肾功能的改变。肾组织分别进行常规苏木素伊红(HE)、磷钨酸苏木素(PTAH)、六胺银-苏木素伊红复合染色(PAM-HE),免疫荧光、光镜观察。结果:静脉注射肾毒血清(NTS)后,模型组大鼠肾功能有明显损伤,表现为血肌酐、尿素氮明显升高,尿蛋白阳性。2周时,取肾组织活检,免疫荧光染色,可见IgG沿肾小球毛细血管壁呈连续线形沉积。7周时,光镜观察可见肾小球系膜细胞增生,肾小球硬化和新月体形成,并有蛋白和红细胞管形。肾炎益气液组上述改变明显轻于模型组。结论:肾炎益气液有不同程度的改善肾功能和减轻肾小球病变的作用。     

糖基化终产物对大鼠肾系膜细胞结缔组织生长因子作用的研究

目的：探讨糖基化终产物（AGEs）对大鼠肾系膜细胞结缔组织生长因子(CTGF) mRNA表达及细胞外基质（ECM）合成的影响。方法： 在培养的大鼠肾系膜细胞中，分别加入不同浓度的糖化牛血清白蛋白(AGE-BSA)及牛血清白蛋白(BSA)进行刺激，ELISA法检测培养上清中的纤连蛋白（FN）及Ⅳ型胶原（ColⅣ）含量，RT-PCR检测细胞CTGF mRNA表达。结果： 与加入BSA组比较，AGE-BSA组CTGF mRNA表达明显增强（P<0.01），上清中FN及ColⅣ合成增加（P<0.01）， 与CTGF mRNA表达量之间呈正相关（r=0.83）。 结论： AGEs能明显诱导大鼠肾系膜细胞CTGF mRNA的表达，提示AGEs可能通过上调CTGF mRNA的表达引起ECM积聚。     

Ⅲ型胶原肾小球病的形态学观察

目的了解Ⅲ型胶原肾小球病的形态学改变,并对Ⅲ型胶原可能的细胞来源进行初步探讨。方法对3例肾活检组织进行光镜、免疫荧光、电镜和Ⅰ、Ⅲ、Ⅳ型胶原及d平滑肌肌动蛋白(α-SMA)的免疫组织化学染色(SP法)观察。结果2例患者临床表现为肾病综合征,其中1例伴高血压,第3例表现为肾功能不全和肾性高血压。3例均无肾病家族史。光镜检查可见肾小球基膜内和系膜区弥漫性过碘酸-希夫反应阳性物质沉积,系膜细胞无明显增生。电镜检查在基膜内疏松层和系膜区可见大量胶原纤维沉积,系膜细胞胞膜下平行排列的束状微丝明显增加。免疫组织化学显示这些胶原纤维为Ⅲ型胶原,Ⅰ型和Ⅳ型胶原阴性,同时系膜区多数系膜细胞α-SMA阳性。结论Ⅲ型胶原肾小球病光镜、电镜及免疫组织化学上都有其特殊的病理改变。肾小球内激活的系膜细胞可能是Ⅲ型胶原的来源。     

5/6肾切除大鼠血清瘦素水平变化及其与肾小球硬化关系

目的:观察5/6肾切除大鼠血清瘦素(leptin)水平及其与肾小球硬化指数(GSI)、肾小球局部转化生长因子-β1(TGF-β1)表达、细胞外基质(ECM)的关系。方法:选用SD雄性大鼠14只,其中8只通过5/6肾切除法制造慢性功能衰竭模型,另6只为假手术组作为正常对照。术后第6周末各组大鼠进行血清肌酐(Scr)、尿素氮(BUN)及leptin的测定,处死大鼠,取出肾组织进行病理组织形态学观察,并采用免疫组织化学方法检测肾小球TGF-β1、Ⅳ型胶原(ColⅣ)及纤维连接蛋白(FN)表达。并对血清leptin水平与GSI、TGF-β1、ColⅣ及FN之间的关系进行相关性分析。结果:5/6肾切除大鼠血清瘦素(leptin)水平显著高于假手术组(14.88±1.46ng/mlvs10.84±2.67ng/ml,P<0.01),并与GSI、TGF-β1、ColⅣ及FN呈显著正相关(P<0.01)。结论:高瘦素血症可能是引起肾小球硬化的机理之一。     

肾炎益气液对肾毒血清性肾炎大鼠肾脏超微结构的影响

目的:观察肾毒血清性肾炎大鼠肾组织超微结构变化及肾炎益气液对该变化的影响。方法: 采用大鼠肾毒血清性肾炎模型,常规电镜制片、染色,观察肾组织超微结构的变化。结果: 注射肾毒血清(NTS)后,病理组大鼠电镜下可见明显的系膜细胞增生,系膜基质增多,毛细血管腔闭塞及上皮下、内皮下电子致密物沉积等多种病理损伤性变化。而肾炎益气液组上述病变有所减轻。结论: 肾炎益气液有不同程度减轻肾小球电镜下病变的作用。     

大鼠ATG肾炎和人IgA肾病肾组织中ⅩⅧ型胶原的表达

目的观察ⅩⅧ型胶原在大鼠抗Thy-1系膜增生性肾小球肾炎（ATG）模型和人IgA肾病肾组织中表达，探讨ⅩⅧ型胶原在肾小球肾炎及肾脏硬化中的作用。方法尾静脉注射兔抗Thy-1血清制备大鼠ATG模型；Western blot检测大鼠ATG肾皮质组织中ⅩⅧ型胶原、Ⅳ型胶原蛋白的表达变化；原位杂交确定ⅩⅧ型胶原mRNA在ATG大鼠肾脏中的细胞定位；免疫组化ABC法观察ⅩⅧ型胶原蛋白在人IgA肾病肾组织的分布，并对其染色强度作定量分析。结果大鼠ATG模型中随着病变的进展肾皮质组织中ⅩⅧ型胶原、Ⅳ型胶原蛋白的表达都呈先持续增加而后回落的趋势；原位杂交显示ⅩⅧ型胶原mRNA在ATG大鼠肾组织中的表达细胞呈多样性；免疫组化结果证实ⅩⅧ型胶原是一种基膜胶原蛋白，在人IgA肾病的细胞外基质沉积中表达显著增加。结论ⅩⅧ型胶原是一种基膜成分，肾脏内多种细胞均可表达ⅩⅧ型胶原，其分布与Ⅳ型胶原相似。     

组织蛋白酶B和胱抑素C在糖尿病肾病大鼠肾组织中的表达及意义

目的： 观察糖尿病大鼠肾组织中组织蛋白酶B(CB)和胱抑素C(CC)的表达并探讨其在糖尿病肾病中的可能作用。方法: 将Wistar大鼠分成健康对照组(C组,n=30)和糖尿病组(M组,n=35),糖尿病组给予STZ 55 mg/kg腹腔内注射,72 h后随机血糖>16.7 mmol/L为造模成功。分别在第4、8、16周各处死10只,在处死前留取24 h尿量测24 h尿白蛋白,留取血清标本测血肌酐,留取肾脏标本做免疫组化和实时荧光定量PCR测CC、CB、Ⅳ型胶原(colⅣ)、纤维连接蛋白(FN)的表达情况。结果: C组的各项指标各时点均未见到有显著变化,与第4周相比,第8周M组大鼠内生肌酐清除率(Ccr)和24 h尿白蛋白显著升高(P<0.01),免疫组化和PCR结果显示CB、CC、colⅣ、FN在第8周出现明显上调(P<0.01或P<0.05),CB在16周下调(P<0.01),而CC显著上调(P<0.01)。相关分析显示CB的表达与24 h尿蛋白、colⅣ、FN蛋白表达和mRNA表达呈负相关（P<0.01）。结论: 糖尿病大鼠肾组织存在CB和CC平衡的失调,这可能是引起糖尿病肾病细胞外基质沉积的原因之一。     

肾小管间质α-SMA表达与大鼠肾炎预后的关系

目的:了解肾毒血清肾炎大鼠肾小管间质α-平滑肌肌动蛋白(α-SMA)的表达与肾炎预后的关系及强的松治疗对其影响。方法:复制大鼠加速型肾毒血清肾炎(NTN)模型,随机分为非治疗和强的松治疗组,在肾炎第7、30、60和90d分别测定血清肌酐(Scr),尿蛋白(UP),肾小管间质形态和α-SMA的表达及分析相互关系。结果:肾小管间质α-SMA的表达与Scr,UP和肾小管间质病变相一致,强的松可抑制α-SMA的表达。结论:α-SMA的表达与肾炎病变的发展有密切关系,强的松影响α-SMA的表达,减少间质细胞的异化,在一定程度上改善肾炎的预后。     

抗纤灵、丹参、大黄等药物血清对系膜细胞分泌FN和Ⅳ型胶原的影响

目的：探讨抗纤灵及其主要成分丹参、大黄等对大鼠肾小球系膜细胞分泌FN和Ⅳ型胶原的影响。方法：采用ElisA法观察中药抗纤灵方、丹参、大黄等药物血清对大鼠肾小球系膜细胞分泌FN和Ⅳ型胶原的影响。结果：（1）抗纤灵、丹参、大黄等含药血清对系膜细胞分泌FN的影响。慢性肾衰（5／6肾切除法）模型组大鼠的血清可显著促进体外培养的系膜细胞分泌FN，假手术组大鼠的血清也可增加系膜细胞分泌FN。抗纤灵组、丹参组、     

血管紧张素(1-7)通过抑制中电导钙激活钾离子通道蛋白表达参与肾脏纤维化

目的 探讨血管紧张素（Ang）（1-7）在肾纤维化过程中的保护作用与中电导钙激活钾离子通道（KCa3.1）的关系。 方法 60只雄性小鼠随机分为5组：对照组 (WT)；血管紧张素Ⅱ（Ang Ⅱ）组：皮下注射 Ang Ⅱ ［1.4 mg/(kg.d)］；注射Ang Ⅱ 的同时给予以下药物干预： Ang Ⅱ阻断剂 洛沙坦（Losartan）组：皮下注射 Losartan［40 mg/(kg.d)］； Ang（1-7）组：皮下注射 Ang（1-7）［0.14 mg/(kg.d)］；血管紧张素转化酶2（ACE2）激动剂重氮氨苯脒乙酰甘氨酸盐（DIZE）组：皮下注射DIZE［10 mg/(kg.d)］。连续给药4周后对相关指标进行检测。Masson染色法检测肾组织胶原沉积变化；Western blotting法检测肾组织 Ⅰ 型胶原、Ⅲ 型胶原和 KCa3.1通道蛋白表达的变化。 结果 与对照组相比，Ang Ⅱ组小鼠肾组织内胶原沉积量明显增加（n=12，P<0.01），表明肾纤维化模型复制成功。Ang Ⅱ使肾组织Ⅰ、Ⅲ型胶原合成显著增多（n=6，P<0.01），同时促进了肾组织KCa3.1通道蛋白的表达（P<0.01），而Ang （1-7）及ACE2激活剂 DIZE 的应用抑制了肾组织内胶原沉积量、Ⅰ/Ⅲ型胶原合成及KCa3.1通道蛋白的表达（n=12或6，P<0.01）。结论 Ang （1-7）在肾纤维化过程中发挥保护作用，这一作用可能与其下调肾组织中KCa3.1通道蛋白表达有关。     

Cocaine reduces macrophage killing by inhibiting reactive nitrogen intermediates

The present study describes the inhibition of macrophage-mediated cytotoxicity (MMC) by cocaine and suggests a possible mechanism. Mice (C57BL/6) were injected i.p. with cocaine. At various intervals after exposure to cocaine, peritoneal macrophages (Mф) were removed, cultured in the presence of interferon γ and LPS, then incubated with ⁵¹Cr labeled target cells. A single injection of ⩾10 mg/kg cocaine was sufficient to inhibit cytotoxicity to P815 cells. This inhibition was evident 3 h after exposure to cocaine and could still be demonstrated 24 h later. Since reactive nitrogen intermediates (RNI) have been reported to be one of the major mechanisms by which Mф kill, the amounts of NO₂⁻ produced by Mф from cocaine-injected animals were compared with that produced by equivalent controls. Cocaine reduced the level of NO₂⁻ in a dose-dependent manner which correlated with MMC. There was a significant reduction in NO₂⁻ produced by activated Mф, 3 h after i.p. injection of cocaine but not at 24 h, using ⩾ 5 mg/kg. At 12 h there were differences between Mф from control animals and animals receiving ⩾10 mg/kg cocaine. By 24 h there were no differences between control and cocaine-injected animals even at the highest dose employed (25 mg/kg). These results suggest that cocaine reduces the killing ability of murine Mф through a temporary reduction of RNI.     

大鼠肾组织血小板衍生的生长因子与肾炎发病的关系

用免疫细胞化学方法研究了大鼠肾毒血清肾炎（ｎｅｐｈｒｏｔｏｉｘｃｎｅｐｈｒｉｔｉｓ，ＮＩＮ）发病头７天肾组织中血小板衍生的生长因子（ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄｇｒｏｗｔｈｆａｃｔｏｒ，ＰＤＧＦ）增生细胞核抗原（ｐｒｏｌｉｆｅｒａｔｉｎｃｅｌｌｎｕｃｌｅａｒａｎｉｇｅｎ，ＰＣＮＡ）和单核／巨噬细胞的变化以及检测这些细胞之间及其与肾功能之间的相关性。结果表明，ＮＴＮ大鼠发病第１天，肾小球内未能检     

L-精氨酸对人肾系膜细胞胞外基质产生的影响

目的:探讨L-精氨酸(L-arg)对人肾系膜细胞胞外基质产生的影响及其作用机制。方法:运用放射免疫分析技术及羟-脯氨酸比色法分别检测L-arg作用后系膜细胞上清液中Ⅲ型前胶原与总胶原含量;运用RT-PCR技术检测L-arg对人肾系膜细胞Ⅳ型胶原基因表达的影响。结果:L-arg抑制Ⅲ型前胶原产生,抑制总胶原分泌,并抑制Ⅳ型胶原基因表达。结论:L-arg抑制人肾系膜细胞胞外基质产生,并可能与抑制胶原成分的基因转录有关。     

Participation of the matrix metalloproteinase inhibitor in Thy-1 nephritis

What influence would be shown in Thy‐1 glomerulonephritis when the synthetic matrix metalloproteinase (MMP) inhibitor SI‐27 is administered? Five groups of 80 male Wistar rats were studied: healthy group; treated healthy group; nephritic group; pretreated nephritic group; and post‐treated nephritic group. SI‐27 treatment of nephritic animals was initiated either 2 days before or 2 days after anti‐Thy‐1.1 antibody injection. On days 7, 14, 26 and 42 after disease induction, we examined renal histology, extracellular matrix (ECM) constituent, and MMP activity. SI‐27 treated Thy‐1 groups resulted in significant reduction of glomerular cells including α‐smooth muscle actin (α‐SMA) positive mesangial cells and suppressed expression of type IV collagen at 7 days. Moreover, type I collagen was also decreased by SI‐27 at 42 days. However, glomerular cell numbers did not show any significant changes at 14, 26 and 42 days. In gelatin zymography, the gelatinolytic band for MMP‐9 was expressed in SI‐27 treated Thy‐1 nephritis groups, although it was not expressed in the nephritic group at day 7. However, the expression of MMP‐9 was no longer seen at 14, 26 and 42 days. The bands for an active form of MMP‐2 were expressed throughout the experimental period in the Thy‐1 nephritic groups. These results suggest that MMP plays an important role in the development of Thy‐1 nephritis, and even if the synthetic MMP inhibitor intercepts the initial increase of glomerular cells and matrices, it does not inhibit recovery to normal glomerular capillary structures in Thy‐1 nephritis.     

不同血液净化方法对人肾小管上皮细胞分泌细胞外基质的影响

目的探讨不同血液净化方法前后尿毒症患者血清对人近端肾小管上皮细胞株（HK-2）分泌细胞外基质（ECM）的影响。方法选择慢性肾衰竭维持性透析（MHD）的患者24例,随机分成3组,分别接受常规血液透析（HD）、高通量透析（HFHD）、血液透析滤过（HDF）后稀释法治疗,提取患者透析前后的血清。将透析前后血清分别混匀后培养HK-2细胞。应用碱水解法、ELISA法检测HK-2细胞培养上清中胶原、纤连蛋白（FN）等ECM的蛋白水平。结果HDF组透析后血清使HK-2细胞胶原蛋白和FN分泌减少,与透析前比较,差异有统计学意义（P〈0.01）;而HD、HFHD组透析后与透析前比较,差异无统计学意义（P〉0.05）。HDF组透析前后胶原蛋白及FN分泌的差值与HD、HFHD组比较,差异有统计学意义（P〈0.01）,而HD组透析前后的差值与HFHD组比较,差异无统计学意义（P〉0.05）。结论HDF可能通过清除致肾间质ECM异常积聚的尿毒症毒素和减少透析用水对机体的影响,从而减少HK-2细胞合成胶原和FN等ECM,对肾间质纤维化的发生、发展起一定的抑制作用,而HD、HFHD在此方面的作用不显著。     

丹酚酸B对高糖诱导的肾小球系膜细胞表型转化及细胞外基质分泌的影响

目的:研究丹酚酸对高糖诱导的肾小球系膜细胞表型转化及细胞外基质分泌的影响及其机制。方法:培养人肾小球系膜细胞(HGMCs),随机分为正常对照组、高糖组及高糖+丹酚酸B高、中、低剂量组,高糖组和丹酚酸B各组用含高浓度(33.3 mmol/L)葡萄糖的培养基培养72 h,丹酚酸B各组同时加人相应浓度丹酚酸B共同孵育。Western blot法检测α-平滑肌肌动蛋白(α-SMA)的表达水平,ELISA法检测细胞Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)、纤维连接蛋白(FN)和层粘连蛋白(LN)的分泌水平,Western blot法检测转化生长因子β1(TGF-β1)的表达及Smad2和p38丝裂原活化蛋白激酶(MAPK)的磷酸化水平。结果:高糖孵育72 h后,肾小球系膜细胞α-SMA的蛋白表达水平明显升高,ColⅠ、ColⅢ、FN及LN蛋白的分泌水平显著增加(P 0.01),TGF-β1的表达及Smad2、p38 MAPK的磷酸化水平也明显升高(P 0.01);与丹酚酸B共同孵育可明显降低α-SMA蛋白的表达水平,ColⅠ、ColⅢ、FN和LN的分泌明显减少,TGF-β1的表达及Smad2、p38 MAPK的磷酸化水平显著下降(P 0.01或P 0.05)。结论:丹酚酸B可明显抑制高糖诱导的肾小球系膜细胞表型转化,减少ColⅠ和ColⅢ等细胞外基质分泌,其机制与抑制TGF-β1/Smad信号通路及p38 MAPK活化有关。     

Alterations in extracellular matrix components in transplant glomerulopathy

The distribution pattern of extracellular matrix (ECM) components in transplant glomerulopathy was studied in relation to light microscopic features, actin expression of mesangial cells, and intraglomerular inflammatory cells. Nine cases of mild (group I) and nine cases of severe (group II) transplant glomerulopathy were stained with antisera against fibronectin (FN), tenascin (TN), collagen types III and IV, smooth muscle actin, CD45RO, CD68, and Ki-67 antigen. The composition of ECM was similar in the two groups. The expanded mesangium was diffusely stained by type-IV collagen, FN and TN, and focally and weakly stained by type-III collagen and smooth muscle actin. Type-IV collagen was linearly stained along the capillary walls, imparting a double-contour feature, whereas FN and TN showed granular staining along the capillary walls. CD68 positive cells were increased in severe transplant glomerulopathy, but this increase was not related to ECM deposition. These findings suggest that increased glomerular deposition of normal and abnormal ECM components participate in the evolution of transplant glomerulopathy. Received: 5 October 1999 / Accepted: 17 January 2000     

Nephrotoxic serum nephritis in nude rats: the roles of host immune reactions in the accelerated type.

By immunization with rabbit immunoglobulins and the injection of a subnephritogenic dose of rabbit nephrotoxic serum (NTS), accelerated-type nephrotoxic serum nephritis (NTN) was induced in heterozygous (rnu/+) rats but not in athymic nude (rnu/rnu) rats. By transferring rat antibody against rabbit immunoglobulins, marked proteinuria was induced also in nude rats (202.0 +/- 98.4 mg/day on day 3) as in rnu/+ rats (122.6 +/- 35.3 mg/day on day 3). No marked differences in histological findings could be found between both groups. The most marked increase in the number of intraglomerular infiltrating cells was observed in heterozygous rats indicating that the presence of thymus-derived cells leads to the accumulation of more cells in glomeruli. We conclude that humoral immunity alone is enough to accelerate the pathogenic mechanism which induces glomerular injury with heavy proteinuria in this model.