Endothelin-positive mast cells in porcine renal artery and vein

For a first time the endothelin (ET)-positive mast cells were examined in the wall of kidney renal artery and vein. The specimen's were collected from six 8-month-old Danmark Landrace pigs, immediately after slaughtering. Mast cells immunopositive to ET granules were observed in the wall of both artery and vein. In the renal artery, they were found mostly between the media and the adventitia. Some mast cells were found in the media, next to smooth muscle cells. Relatively few mast cells were found in the intima and between intima and tunica media. In the renal vein a smaller number of mast cells were observed. They showed similar localization as in the renal artery. Immunopositive mast cells were established also close to endothelial cells - mostly between internal elastic membrane and basal membrane of the endothelium. In conclusion, on the basis of obtained results, presumptions for active participation of ET (most probably mainly ET-1) in the motility of the vessels' smooth muscle and for stimulation of nitric oxide release from the intimal endothelial cells were made.     

Activation of complement system in kidney after ketoprofen-induced kidney injury in sheep

Background

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammatory pain in humans and animals. An overdose of an NSAID is nephrotoxic and can lead to acute kidney injury (AKI). Complement activation occurs in several types of renal disorders with proteinuria. The aim of this study was to investigate whether complement system becomes activated in kidneys after a high dose of NSAID. Kidney tissue and urine samples were collected from six sheep with ketoprofen-induced AKI and from six healthy control sheep. The localization of complement proteins in kidney tissue was carried out using immunohistochemical stainings, and excretion of C3 was tested by immunoblotting.

Results

The complement system was found to become activated in the kidney tissue as demonstrated by positive immunostaining for C1q, C3c, C4c, C5, C9 and factor H and by Western blotting analysis of C3 activation products in urine samples in sheep with AKI.

Conclusions

Our results thus suggest that the alternative complement pathway is activated, and it may contribute to the acute tubular injury seen in the kidneys of NSAID-induced AKI sheep. Inhibition of complement activation may serve as potential therapeutic target for intervention in drug-induced AKI.     

Localization of potential binding sites for the edema disease verotoxin (VT2e) in pigs.

The purpose of this study was to identify organs and cells to which the edema disease verotoxin (VT2e) could bind in pigs. Frozen 4-5 microns thick sections of organs usually affected in edema disease (colon, spinal cord, cerebellum and eyelid) and organs not usually affected (liver, ileum) from two 5- to 6-week-old weaned pigs were permeabilized with acetone, then exposed to VT2e. Unbound VT2e was removed by washing and bound VT2e was detected by immunohistochemistry. In the eyelid, double-label immunofluorescence was used to identify the cells to which VT2e bound. VT2e was shown to bind to all six organs that were examined. The toxin bound to arteries in all organs, to veins in all organs except the liver, and to enterocytes in the ileal crypts. Double labelling of eyelid with monoclonal antibodies specific for von Willebrand factor or alpha-smooth actin and VT2e showed that the toxin bound to endothelial and vascular smooth muscle cells. The binding of VT2e to endothelium is consistent with findings for other verotoxins but binding to vascular smooth muscle has not been reported for other verotoxins. It is concluded that i) factors other than the presence of receptors for VT2e influence the development of lesions in edema disease, and ii) smooth muscle necrosis, which is characteristic of the vascular lesions in edema disease, may be due to a direct action of toxin on smooth muscle cells.     

发情周期不同阶段牦牛子宫中黄体生成素受体的表达变化

为研究发情周期不同阶段牦牛子宫中黄体生成索受体(LHR)的定位及表达变化,笔者利用免疫组织化学SP法分别检测发情期、发情后期、间情期和发情前期牦牛子宫中LHR的表达,并进行光密度值分析.结果表明,LHR免疫阳性产物在牦牛子宫腺上皮细胞、基质细胞、血管内皮细胞、血管平滑肌细胞和肌层平滑肌细胞中均有表达;腺上皮细胞、基质细胞和肌层平滑肌细胞中LHR在发情前期和发情期表达最弱,发情后期表达增加,间情期表达最强(P＜0.05);子宫内膜血管平滑肌细胞中LHR的表达在发情期最强,间情期最弱(P＜0.05);血管内皮中LHR在发情期和发情前期表达很强,发情后期和间情期显著下降(P＜0.05).结果表明LHR参与了发情周期不同阶段牦牛子宫功能变化的调控.     

Neuromuscular and vascular hamartoma of the small intestine in an F344 rat

An intestinal mass was found in the border area of the jejunum and ileum of a 110-week-old male F344 rat. Histopathologically, the mass protruded into the lumen and was covered with intestinal epithelium, exhibiting a normal architecture. The lesion was located in the submucosa and consisted of loose connective tissue, smooth muscle, scattered ganglion cells, and blood vessels of various sizes. Although these components showed an irregular and disordered structure, no cellular atypia, increased proliferation activity, or invasive growth to adjacent tissues were detected. Immunohistochemical analyses revealed that smooth muscle, ganglion, and endothelial cells were positive for α-smooth muscle actin and vimentin, S-100, and CD34 and von Willebrand factor, respectively, indicating maturation of these cells. Thus, the mass was diagnosed as a neuromuscular and vascular hamartoma of the small intestine. To the best of our knowledge, this is the first report of this type of lesion in rodents.     

Isolation and characterization of canine tumor endothelial cells

The present study involved the isolation and characterization of canine tumor endothelial cells (TECs) from 2 malignancies. TECs were isolated using magnetic cell sorting following FITC labeling with UEA1 lectin, and they were characterized by measuring genetic and histopathological endothelial markers. Isolated TECs exhibited a cobblestone-like morphology and expressed both vascular endothelial growth factor receptor 2 (VEGFR2) and Von Willebrand factor (vWF). Further, both TECs and tumor cells derived from a seminoma exhibited increased C-X-C chemokine receptor type 7 (CXCR7) expression. However, CXCR7 expression was not detected in TECs and tumor cells derived from a hepatocellular carcinoma. Understanding TEC specific traits may be important in the development of more efficacious anti-angiogenic therapies that do not induce adverse effects.     

Mechanisms of acetylcholine-induced vasorelaxation in high K+-stimulated rabbit renal arteries

To characterize the mechanisms of acetylcholine (ACh)-induced vasorelaxation in rabbit renal arteries precontracted with high K+ (100 mM), muscle tension and cytosolic free Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded arterial strips. In the artery with endothelium, high K+ increased both [Ca2+]i and muscle tension. Addition of ACh (10 microM) during high-K+ induced contraction significantly relaxed the muscle and induced additional increase in [Ca2+]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM). ACh increased [Ca2+]i without relaxing the muscle. In the artery without endothelium, high K+ increased both [Ca2+]i and muscle tension although ACh was ineffective, suggesting that ACh acts selectively on endothelium to increase [Ca2+]i. 4-DAMP (10 nM) or atropine (0.1 microM) abolished the ACh-induced increase in [Ca2+]i and relaxation. However, pirenzepine (0.1 microM), AF-DX 116 (1 microM) and tropicamide (1 microM) were ineffective. The ACh-induced increase of [Ca2+li and vasorelaxation was significantly reduced by 3 microM gadolinium, 10 microM lanthanum or 10 microM SKF 96365. These results suggest that, in rabbit renal artery, ACh-evoked relaxation of 100 mM K+-induced contractions is mediated by the release of endothelial NO. ACh may stimulates the M3 subtype of muscarinic receptor in the endothelial cells, resulting in the opening of the nonselective cation channels followed by an increase of [Ca2+]i and stimulation of NO synthase.     

Early arterial injury-induced myointimal proliferation in canine pulmonary arteries

Transmission electron microscopy was used to study the early vascular response induced by arterial damage with heartworm infection. The pulmonary arteries were examined in dogs 4 days after experimental transplantation of 6 to 8 Dirofilaria immitis adults. Evan's blue dye was given IV and followed in 60 minutes by perfusion fixation with 1% glutaraldehyde. Endothelial cell junctions were disrupted and rounded endothelial cells were observed. Macrophages and neutrophils adhered to abnormal endothelial cells. Focal areas had platelet aggregates adhered to exposed subendothelial structures. Platelets were activated and degranulated. Areas of the internal elastic lamina appeared to be disrupted, and smooth muscle cell processes from the tunica media extended through these regions of disruption. Smooth muscle cells were oriented toward the arterial lumen and some had migrated to the surface. These findings are compatible with the response to injury theory of myointimal proliferation.     

PRRSV感染仔猪肾组织的超微病理变化分析

猪繁殖与呼吸综合征病毒（PRRSV）是能引起仔猪高死亡率的疾病,给养猪业带来极大的经济损失。本研究对PRRSV感染仔猪肾组织的病毒定位及产生的超微病理变化进行观察,以期了解PRRSV对肾组织细胞以及血液循环的影响。主要采用透射电镜技术结合血清生化检测、免疫组织化学染色（IHC）,对仔猪感染PRRSV后肾组织进行超微病理学观察及分析。结果发现,PRRSV感染后引起肾组织损伤,病毒主要分布于肾小球、坏死的肾小管上皮细胞胞质及巨噬细胞胞质内;肾小球内足细胞足突广泛融合或微绒毛化,内皮细胞肿胀;肾小管上皮细胞线粒体肿胀、嵴断裂、溶解;间质炎性细胞浸润。PRRSV通过巨噬细胞内复制随血液扩散至肾组织,引起损伤;根据透射电镜观察结果,足细胞足突融合,肾小球血管内皮细胞损伤引起微血栓形成,说明PRRSV感染仔猪后影响了肾小球滤过率,同时,对肾血液微循环系统造成损伤。本研究为阐明PRRSV的感染致病机制提供理论基础。     

The use of novel lymphatic endothelial cell‐specific immunohistochemical markers to differentiate cutaneous angiosarcomas in dogs

Lymphangiosarcomas are uncommon vascular neoplasms that arise from lymphatic endothelial cells (LECs). They efface and replace normal subcutaneous tissue and are characterised by arborising, vascular channels lined by a single layer of pleomorphic endothelial cells and a paucity of erythrocytes. Lymphangiosarcomas are architecturally similar to hemangiosarcomas, a common malignancy of vascular origin arising from blood vascular endothelial cells. Common immunohistochemical markers for vascular endothelium, such as Factor VIII‐related antigen (F8RA) and CD31, have traditionally been used to confirm the diagnosis of tumours of vascular origin. However, these markers fail to differentiate between lymphangiosarcoma and hemangiosarcoma, which often show overlapping morphologic features, disparate clinical behaviour and require different treatment modalities. Here we describe the use of two novel LEC‐specific markers, lymphatic vessel endothelial receptor‐1 (LYVE‐1) and prospero‐related homeobox gene‐1 (PROX‐1), to further differentiate between vascular tumours of lymphatic (lymphangiosarcoma) and blood (hemangiosarcoma) endothelial cell origin in the dog.     

K_(Ca)3.1通道与血管功能的研究进展

研究表明,在促进血管疾病发展的众多细胞类型中,如T细胞、B细胞、内皮细胞、成纤维细胞、巨噬细胞以及去分化的平滑肌细胞,KCa3.1都起到了一定的作用。生理上,KCa3.1已被证明在乙酰胆碱及内皮源性超极化因子(EDHF)介导下发生超极化,从而发挥控制血压的作用。病理上,KCa3.1异常则可导致血管平滑肌细胞增殖迁移。因此,从KCa3.1在内皮细胞及平滑肌细胞两方面对其作用作一总结,并对KCa3.1通道的研究进展进行综述。     

Endothelial–platelet interactions in influenza-induced pneumonia: A potential therapeutic target

Every year, influenza viruses spread around the world, infecting the respiratory systems of countless humans and animals, causing illness and even death. Severe influenza infection is associated with pulmonary epithelial damage and endothelial dysfunction leading to acute lung injury (ALI). There is evidence that an aggressive cytokine storm and cell damage in lung capillaries as well as endothelial/platelet interactions contribute to vascular leakage, pro-thrombotic milieu and infiltration of immune effector cells. To date, treatments for ALI caused by influenza are limited to antiviral drugs, active ventilation or further symptomatic treatments. In this review, we summarize the mechanisms of influenza-mediated pathogenesis, permissive animal models and histopathological changes of lung tissue in both mice and men and compare it with histological and electron microscopic data from our own group. We highlight the molecular and cellular interactions between pulmonary endothelium and platelets in homeostasis and influenza-induced pathogenesis. Finally, we discuss novel therapeutic targets on platelets/endothelial interaction to reduce or resolve ALI.     

Urinary Biomarkers for Acute Kidney Injury in Dogs

Routinely, kidney dysfunction and decreased glomerular filtration rate (GFR) are diagnosed by the evaluation of changes in the serum creatinine (SCr) and blood urea nitrogen (BUN) concentrations. However, neither of these tests is sensitive or specific enough for the early diagnosis of impaired kidney function because they are both affected by other renal and nonrenal factors. Furthermore, kidney injury can be present in the absence of kidney dysfunction. Renal reserve enables normal GFR even when nephrons are damaged. Renal biomarkers, especially those present in urine, may be useful for the study of both acute and chronic nephropathies. The aim of this review is to describe the current status of urinary biomarkers as diagnostic tools for kidney injury in dogs with particular focus on acute kidney injury (AKI). The International Renal Interest Society (IRIS) canine AKI grading system and the implementation of urinary biomarkers in this system also are discussed. The discovery of novel urinary biomarkers has emerged from hypotheses about the pathophysiology of kidney injury, but few proteomic urine screening approaches have been described in dogs. Lack of standardization of biomarker assays further complicates the comparison of novel canine urinary biomarker validation results among studies. Future research should focus on novel biomarkers of renal origin and evaluate promising biomarkers in different clinical conditions. Validation of selected urinary biomarkers in the diagnosis of canine kidney diseases must include dogs with both renal and nonrenal diseases to evaluate their sensitivity, specificity as well as their negative and positive predictive values.     

酶消化法分离培养肉鸡肺动脉平滑肌细胞

试验旨在探讨胶原酶消化法分离并培养肉鸡肺动脉平滑肌细胞(pul monary artery smooth muscle cell,PASMC)的方法。将1～7日龄雏鸡的肺动脉中膜剪成小于1 mm2的组织块,0.1%Ⅱ型胶原酶消化分离PASMC并进行原代培养和传代培养。倒置显微镜下观察培养细胞的形态,对培养细胞进行HE染色和光学显微镜观察,并采用抗α-肌动蛋白单克隆抗体(α-Smooth muscle actin)免疫组化染色法检测细胞胞浆内α-肌动蛋白的表达,用以鉴定所培养的细胞。结果表明,倒置显微镜下细胞为长梭形,呈典型的"峰—谷"状,未见明显异型细胞。α-actin胞浆染色阳性细胞数占总细胞数的97%以上。因此,胶原酶消化法可用于分离培养肉鸡PASMC,且有方法简单,成活细胞数多、传代周期短的特点,可用于肉鸡心血管疾病如肺动脉高压和肺血管重构的研究工作。     

Characterization of muscarinic receptor subtype mediating contraction and relaxation in equine coronary artery in vitro

In coronary arterial rings isolated from horses, 10-^-8-10^-6 mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with I,-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of Ach 10-¹⁴-10-⁸ mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine ≥ parafluoro-hexahydrosiladiphenidol (pFHHSiD) > methoctramine. ACh-induced relaxation in the rings with endothelium was inhibited by LNAG or MB, and by each selective antagonist in the following order of potency: 4-DAMP < pFHHSiD ≥ pirenzepine ≥ methoctramine. These results suggest that the ACh-induced contraction and relaxation in equine coronary arteries are mediated mainly by an M₃-receptor located on the smooth muscle cells and endothelial cells, respectively, and that the stimulation of the M₃-receptor on the endothelial cells liberates nitric oxide.     

Kidney-derived proteins in urine as biomarkers of induced acute kidney injury in sheep

Acute kidney injury (AKI) is a life-threatening condition for which an early diagnosis is problematic. The aim of the present study was to identify kidney-derived urinary proteins specific to AKI in sheep. AKI was induced in six sheep by an overdose of ketoprofen. Six untreated sheep served as controls. Urine samples were collected for up to 24 h after drug administration and pooled according to time and treatment. Tissue samples from kidney were taken immediately after euthanasia. Urinary proteins were separated by two-dimensional gel electrophoresis (2DE) and the proteins of interest were identified by mass spectrometry. Calbindin-D28k, retinol-binding protein 4 and CD1d were identified in ketoprofen-treated sheep, but not in controls. In addition, calbindin-D28k and CD1d were localized in kidney tissues by immunohistochemical staining. These preliminary results suggest that urinary calbindin-D28k and CD1d represent potential useful biomarkers of AKI, at least in sheep.     

Spontaneous well-differentiated pancreatic islet cell carcinoma with vascular invasion in a male F344 rat

This case report describes a case of spontaneous pancreatic islet cell carcinoma with vascular invasion in a 110-week-old male F344 rat. Histologically, a pancreatic nodule consisting of tumor cells and many blood-rich vessels, and covered with a fibrous capsule showed local invasion in the capsule and adjacent acinar tissues, encircling a large duct-like structure (DS). The tumor was composed of well-differentiated tumor cells resembling normal pancreatic islet cells, which had small round nuclei and eosinophilic cytoplasm. Mitotic figures were rare. Immunohistochemistry revealed that the tumor cells were positive for insulin. Although endothelial cells were not detected, the DS wall showed cells positive for α-smooth muscle actin and elastic fibers, suggesting that the DS is the pancreatic artery. This is a rare case of islet cell carcinoma consisting of well-differentiated tumor cells with invasion of the pancreatic artery in a rat.     

Labelling of rat endothelial cells with antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen-1 (RECA-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), OX-43 and zonula occludentes-1 (ZO-1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA-1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM-1, ICAM-1 and OX-43 was negative in both adult and embryonic endothelial cells. ZO-1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5-day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5-day yolk sac vessels stained weakly, staining gradually increased in 13.5-, 15.5- and 17.5-day-old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.     

电针疗法对AKI犬的肾功能、钙磷代谢、抗氧化能力以及NRF2信号通路的影响

急性肾损伤(acute kidney injury, AKI)目前已成为犬最主要的一种肾系疾病,尤其在老年犬中发病率逐年上升。近年来,针刺疗法在宠物临床已得到广泛的应用,并逐渐从治疗瘫痪等外科疾病向治疗宠物内科疾病的方向发展。本研究以腺嘌呤为造模药物建立急性肾损伤犬模型,采用电针疗法进行治疗。研究电针疗法对于犬的肾功能、钙磷代谢、抗氧化能力以及对于NRF2通路相关蛋白的影响,探究电针疗法对犬的急性肾损伤的治疗作用。选取24只3～4 kg健康比格犬,随机分为对照组、造模组、常规治疗组、电针干预组、电针治疗组和联合治疗组。第1～15天为造模期,第16～30天为治疗期。试验结束后检测尿比重、血清中尿素氮(BUN)、肌酐(CREA)、尿酸(UA)、钙(Ca²⁺)和磷(P³⁺)的变化;影像诊断X光检测肾变化情况;检测血清及肾组织的超氧化物歧化酶(SOD)和丙二醛(MDA);病理组织学观察各组试验犬肾组织病变的严重程度;采用qRT-PCR和Western blot方法检测各组试验犬肾组织中与抗氧化相关基因和蛋白的表达情况;用免疫组织化学的方法检测肾组织中...