Effects of Hyperosmolality on Expression of Urea Transporter A2 and Aquaporin 2 in Mouse Medullary Collecting Duct Cells

In this study,the effects of hyperosmolality on the expression of urea transporter A2 (UTA2) and aquaporin 2 (AQP2) were investigated in transfected immortalized mouse medullary collecting duct (mIMCD3) cell line.AQP2-GFP-pCMV6 and UTA2-GFP-pCMV6 plasmids were stably transfected into mIMCD3 cells respectively.Transfected mIMCD3 and control cells were cultured in different hy-pertonic media,which were made by NaCl alone,urea alone,or an equiosmolar mixture of NaCl and urea.The mRNA and protein expression of AQP2 was elevated by the stimulation of NaCl alone,urea alone and NaCl plus urea in AQP2-mIMCD3 cells;whereas NaCl alone and NaCl plus urea rather than urea alone increased the mRNA and protein expression of UTA2 in UTA2-mIMCD3 cells,and all the expression presented an osmolality-dependent manner.Moreover,the mRNA and protein expression of UTA2 rather than AQP2 was found to be synergistically up-regulated by a combination of NaCl and urea in mIMCD3 cells.It is concluded that NaCl and urea synergistically induce the expression of UTA2 rather than AQP2 in mIMCD3 cells,and hyperosmolality probably mediates the expression of AQP2 and UTA2 through different mechanisms.

Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

2型糖尿病阴虚证与尿AQP2、AQP7表达的相关性研究

目的观察2型糖尿病阴虚证临床特征,明确患者尿中水通道蛋白-2(aquaporin-2,AQP2)、水通道蛋白-7(aquaporin-7,AQP7)表达特点及相关因素对其的影响,以利于进一步探索2型糖尿病阴虚证病证结合证候特点。方法制定2型糖尿病临床观察表、2型糖尿病阴虚证辨证标准量表以及中医证候诊断标准量表,将2021年3月—2021年12月北京中医药大学东直门医院肾病内分泌科住院部34例2型糖尿病患者分为阴虚证组(观察组)和阳虚证组(对照组)。采集两组患者血生化标本及清晨第一次全部尿液并采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测尿AQP2、AQP7的表达水平。对病例的性别、年龄、病程等多因素进行校正,分析两组尿AQP2、AQP7的差异。结果经校正后,2型糖尿病阴虚证患者尿AQP2水平明显低于阳虚证患者,MD(95%CI)为-64.23(-93.59,-34.87)(P<0.01),而尿AQP7水平低于阳虚证患者,MD(95%CI)为-3.98(-9.77,1.80)(P>0.05)。结论该研究所纳入2型糖尿病阴虚证患者尿AQP2指标表达低于阳虚证患者,提示AQP2可能是2型糖尿病阴虚证特异性生物标志物。

血清及尿β_(2)微球蛋白、视黄醇结合蛋白、水通道蛋白-2与肾盂输尿管连接部梗阻致小儿肾积水严重程度的关系研究

目的探讨血清及尿β_(2)微球蛋白(β_(2)-MG)、视黄醇结合蛋白(RBP)、水通道蛋白-2(AQP2)与肾盂输尿管连接部梗阻(UPJO)致小儿肾积水严重程度的关系。方法选取南宁市第六人民医院泌尿外科接受肾盂输尿管梗阻手术治疗的60例肾积水患儿为肾积水组,根据肾积水严重程度分为轻度组(n=13)、中度组(n=22)和重度组(n=25),另外选取同期该院接受体检的60例健康儿童为对照组。比较对照组和肾积水组不同严重程度患儿血清及尿β_(2)-MG、RBP、AQP2水平;分析β_(2)-MG、RBP、AQP2水平与超声参数[肾盂前后径(APD)、肾实质厚度(PT)]的相关性;采用受试者工作特征(ROC)曲线分析血清及尿β_(2)-MG、RBP、AQP2对中重度肾积水的诊断效能。结果肾积水组血清及尿β_(2)-MG、RBP水平均高于对照组,血清及尿AQP2水平均低于对照组,差异有统计学意义(P<0.05)。肾积水患儿血清β_(2)-MG、尿β_(2)-MG、尿RBP水平和APD水平由低到高为轻度组、中度组、重度组,尿AQP2水平和PT由高到低为轻度组、中度组、重度组,差异均有统计学意义(P<0.05)。轻度组血清RBP水平低于中度组和重度组,差异有统计学意义(P<0.05)。肾积水患儿血清β_(2)-MG、尿β_(2)-MG、尿RBP水平与APD均呈正相关,与PT均呈负相关(P<0.05);尿AQP2水平与APD呈负相关,与PT呈正相关(P<0.05)。ROC曲线分析结果显示,血清β_(2)-MG、尿β_(2)-MG、尿RBP、尿AQP2检测对中重度肾积水均具有一定诊断价值,曲线下面积(AUC)分别为0.652、0.688、0.859、0.680,尿RBP诊断中重度肾积水的AUC显著大于其余指标(P<0.05)。结论血清β_(2)-MG、尿β_(2)-MG、尿RBP、尿AQP2与UPJO致小儿肾积水严重程度相关,尿RBP对中重度肾积水的诊断效能优于其他指标。

葡萄籽原花青素提取物对心肌梗死大鼠肾素-血管紧张素系统及AQP2蛋白表达的影响

目的:探讨葡萄籽原花青素提取物(GSPE)对心肌梗死大鼠肾素血管紧张素-系统及水通道蛋白2(AQP2)表达的影响。方法:将55只无特定病原体(SPF)级Sprague-Dawley(SD)大鼠按照随机数字表法分为健康组、模型组、辛伐他汀组、GSPE低剂量组及GSPE高剂量组,每组11只。除健康组外其余大鼠均采用冠状动脉结扎法制备心肌梗死模型,建模成功后,GSPE低剂量组、高剂量组大鼠分别灌胃100 mg/kg、400 mg/kg葡萄籽原花青素溶液,辛伐他汀组灌胃40 mg辛伐他汀溶液。苏木精-伊红(HE)染色后观察心肌组织形态;末端脱氧核苷酸转移酶介导的原位缺口末端转移酶标记法(TUNEL)检测心肌细胞凋亡;超声心动图监测心功能;放射免疫法检测血管紧张素Ⅱ(AngⅡ)、血浆肾素活性(PRA)、醛固酮水平;免疫印迹法检测心肌组织中AQP2、B细胞淋巴瘤/白血病2号基因(Bcl-2)及半胱天冬氨酸蛋白酶-3(Caspase-3)蛋白水平。结果:与健康组比较,模型组大鼠左心室收缩末期内径(LVESD)、左心室舒张末期内径(LVEDD)升高,左心室射血分数(LVEF)和左心室短轴缩短分数(LVFS)降低(P<0.05);与模型组比较,GSPE低剂量组、GSPE高剂量组及辛伐他汀组LVEDD、LVESD降低,LVEF、LVFS升高(P<0.05),GSPE低剂量组、GSPE高剂量组间比较差异有统计学意义,而GSPE高剂量组与辛伐他汀组比较差异无统计学意义(P>0.05)。与健康组比较,模型组大鼠AngⅡ、PRA、醛固酮升高(P<0.05);与模型组比较,GSPE低剂量组、GSPE高剂量组及辛伐他汀组AngⅡ、PRA、醛固酮降低(P<0.05)。健康组、模型组、GSPE低剂量组、GSPE高剂量组及辛伐他汀组的心肌细胞凋亡率分别为(5.11±0.33)%、(46.22±3.97)%、(28.46±3.77)%、(15.42±2.33)%及(16.34±2.57)%,各组比较差异有统计学意义(P<0.05)。与健康组比较,模型组大鼠心肌组织中AQP2、Caspase-3蛋白水平升高,Bcl-2蛋白水平降低(P<0.05);与模型组比较,GSPE低剂量组、GSPE高剂量组、辛代他汀组大鼠心肌组织中AQP2、Caspase-3蛋白水平降低,Bcl-2蛋白水平升高(P<0.05)。结论:GSPE对心肌梗死有保护作用,可改善心功能水平,减少心肌细胞凋亡,其作用机制可能与调控AQP2蛋白表达有关。

充血性心力衰竭水潴留的机制——Aquaporin2水通道蛋白的作用

水潴留是充血性心力衰竭的重要病理生理表现。长期以来人们对心力衰竭时水重吸收增加的肾脏机制所知甚少。Aquaporin水通道 (AQP)家族的发现是水通道生物学研究的一个新的里程碑。研究发现充血性心力衰竭时肾脏AQP2水通道基因mRNA和蛋白的表达明显上调 ;AQP2的上调主要由血管加压素所介导 ,V2受体拮抗剂可显著降低此上调 ;在充血性心力衰竭时肾脏AQP2表达的改变是选择性的 ,不伴有肾脏AQP1和AQP3蛋白表达的增加。证实AQP2水通道是充血性心力衰竭时调节水潴留的关键性蛋白质 。

高心排心力衰竭大白鼠肾脏Aquaporin2水通道蛋白基因的表达

目的 :研究高心排心力衰竭大白鼠 Aquaporin2基因的表达及精氨酸血管加压素 ( AVP)受体拮抗剂的治疗作用。方法 :SD大白鼠分别行腹腔动脉 -下腔静脉分流术 ,建立高心排心力衰竭动物模型。术后随机分为 A- C分流对照组、A- C分流及 V1受体拮抗剂治疗组、A- C分流及 V2受体拮抗剂组 ,和 A- C分流及 V1/ V2受体拮抗剂治疗组。每周测定尿量 ,尿渗透压 ,4周后测定 AVP浓度 ,同时做 AQP2基因蛋白表达的检测。结果 :A- C分流大白鼠 4周后出现 AVP水平升高 ( P <0 .0 1) ,同时伴有 AQP2基因蛋白表达增加 ;AVPV2受体拮抗剂能抑制 A- C分流过程中 AVP的升高及 AQP2基因蛋白的过度表达。结论 :A- C分流大白鼠 AQP2基因蛋白表达增加 ,其与AVP水平相关 ;AVP V2受体拮抗剂能够改善心力衰竭时的水钠潴留 ,为开发 AVP V2受体拮抗剂这类新型利尿药物提供了理论基础。

Effect of Micardis on the Expression of Renal Medulla Aquaporin-2 in Diabetic Mice

In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 （AQP2） was investigated in the renal medullary collecting duct of mice with diabetic nephropathy （DN）. Mice were divided into three groups： normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group （2.39±0.11 vs 3.16±0.16, P〈0.05）. Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system （RAS） is implicated in the pathogenesis of DN by regulating the expression of AQP2.

电针对膜迷路积水豚鼠耳蜗V2R、AQP2、ENaC-α表达的影响

目的探讨电针对血管加压素(arginine vasopressin,AVP)诱导的美尼尔综合征膜迷路积水豚鼠耳蜗血管加压素受体2(vasopressin type 2 receptor,V2R)、水通道蛋白2(aquaporin 2,AQP2)、上皮细胞钠离子通道蛋白-α(epithelial sodium channel-α,ENaC-α)表达的影响,初步阐明电针干预美尼尔综合征膜迷路积水机制。方法白色红目豚鼠分为正常组、模型组、电针刺组,每组12只。采用AVP腹腔注射建立膜迷路积水模型。电针刺组给予“百会”、左侧“听宫”电针治疗,20min/次,每日1次,共治疗10日。治疗结束后每组6只豚鼠采用HE染色法观察耳蜗积水程度,每组6只豚鼠采用Western blot法检测左侧耳蜗V2R、AQP2、ENaC-α蛋白表达水平。结果与正常组比较,模型组耳蜗积水程度增加(P<0.01),与模型组比较,电针刺组耳蜗积水程度降低(P<0.01)。与正常组比较,模型组耳蜗V2R、AQP2、ENaC-α蛋白表达增加(P<0.01),与模型组比较,电针刺组耳蜗V2R、AQP2、ENaC-α蛋白表达减弱(P<0.01)。结论电针可改善膜迷路积水豚鼠耳蜗积水程度,其机制可能与下调耳蜗V2R、AQP2、ENaC-α蛋白表达有关。

Downregulation of Aquaporin 4 Expression through Extracellular Signal-regulated Kinases1/2 Activation in Cultured Astrocytes Following Scratch-injury

Objective To investigate the role of extracellular signal-regulated kinase1/2（ERK1/2） pathway in the regulation of aquaporin 4（AQP4） expression in cultured astrocytes after scratch-injury. Methods The scratch-injury model was produced in cultured astrocytes of rat by a 10-μL plastic pipette tip. The morphological changes of astrocytes and lactate dehydrogenase（LDH） leakages were observed to assess the degree of scratch-injury. AQP4 expression was detected by immunofluorescence staining and Western blot, and phosphorylated-ERK1/2（p-ERK1/2） expression was determined by Western blot. To explore the effect of ERK1/2 pathway on AQP4 expression in scratch-injured astrocytes, 10 μmol/L U0126（ERK1/2 inhibitor） was incubated in the medium at 30 min before the scratch-injury in some groups. Results Increases in LDH leakage were observed at 1, 12, and 24 h after scratch-injury, and AQP4 expression was reduced simultaneously. Decrease in AQP4 expression was associated with a significant increase in ERK1/2 activation. Furthermore, pretreatment with U0126 blocked both ERK1/2 activation and decrease in AQP4 expression induced by scratch-injury. Conclusion These results indicate that ERK1/2 pathway down-regulates AQP4 expression in scratch-injured astrocytes, and ERK1/2 pathway might be a novel therapeutic target in reversing the effects of astrocytes that contribute to traumatic brain edema.

Tolvaptan regulates aquaporin-2 and fecal water in cirrhotic rats with ascites

AIM: To investigate the role of tolvaptan in regulating aquaporin(AQP)-2 expression and fecal water content in cirrhotic rats with ascites.METHODS: Cirrhosis with ascites was induced in rats by repetitive dorsal injection of CCl4 for 14 wk. In total, 84 cirrhotic rats with ascites divided into three groups(vehicle, 3 mg/kg and 5 mg/kg tolvaptan), and then further divided into five subgroups(days 1, 2, 3, 4, and 5). Blood samples were obtained to measure vasopressin and sodium concentrations. Rats were killed and colonic mucosa was scraped for analysis of protein expression and AQP-2 transcriptional level. The whole layer was fixed for hematoxylin&eosin(HE) staining and feces were collected for determination of fecal water content. CONCLUSION: Compared with vehicle, vasopressin decreased significantly in the tolvaptan groups from day 2 to a similar level in each treatment group. AQP-2 showed significant upregulation in cirrhotic rats with ascites compared with an untreated control group(100% ± 22.9% vs 22.2% ± 10.23%, P < 0.01). After administration of tolvaptan, AQP-2 expression began to decrease significantly from day 2 in each treatment group, but no significant difference was finally found between the treatment groups. Fecal water content inthe distal colon was increased by 5 mg/kg tolvaptan on day 1(66.8% ± 9.3% vs 41.4% ± 6.3%, in the vehicle group, P < 0.05). Fecal water content returned to baseline at day 4 at the latest in both treatment groups, and did not correspond to the change in AQP-2 expression. HE staining of the colonic mucosa showed no mucosal damage related to tolvaptan.CONCLUSION: Upregulation of AQP-2 in the distal colon is found in cirrhotic rats with ascites. Tolvaptan inhibits its expression and may decrease water reabsorption and induce diarrhea.

Aquaporin 1 Facilitated Hepatocellular Carcinoma SMMC7221 Cell Migration Associated with Water Permeability

The authors investigated the regulation of human aquaporin I（hAQP1） and the involvement of aquaporin 1 （AQP 1） in the migration of human hepatocellular carcinoma SMMC-7221 cells using RNA intereference technology Firstly, two short hairpin RNA（shRNA） constructs in PBSU6 vector were reconstructed and their knockdown effects were identified in SMMC-7221 cells. Next, the involvement of endogenous hAQP1 in regulating the migration of SMMC-7221 cells was investigated via siRNA technology. HAQPI-shRNA can specifically inhibit AQP1 dependent osmotic water permeability. Meanwhile the migration of SMMC-7221 cells was inhibited remarkably after silencing AQP1 by performing transwell cell migration assay and in vitro wound healing assay. Furthermore, in the presence of an inhibitor HgCl2, the water permeability of the cell membrane was remarkably decreased, the expression of AQP1 was upregulated after HgCla treatment and the cell movement was decreased at the moment. Increased AQP1 cannot attenuate cell migration ability when cell membrane loses its water permeability function. This demonstrates that the cell migration was remarkably related to the transporting water function of cell membrane.

Influence of Sanao Tang on urine volume and expression of aquaporin 2 in rats with respiratory function impairment induced by passive smoking and lipopolysaccharide

OBJECTIVE: To investigate the effect of Sanao Tang(SAT) on urine volume and the expression of aquaporin-2(AQP2) in rats with lung dysfunction induced by passive smoking and lipopolysaccharide.METHODS: Totally 45 healthy Specific pathogen Free Wistar Rats were randomized into 3 groups:normal control group, model group and SAT group.A rat model of respiratory dysfunction induced by exposure to cigarette smoking and lipopolysaccharide(LPS). Lavage of decoction of the Chinese medicine was performed for rats in the SAT group. Anires 2005 System was used to analyze the pulmonary function. Urine of rats was collected through metabolism cage method. Enzyme-linked immunosorbent assay(ELISA) was used to determine content of antidiuretic hormone(ADH), angiotensin Ⅱ(AngⅡ), atrial natriuretic factor(ANP), endothelin 1(ET-1), nitric oxide(NO) and prostaglandin E2(PGE2) in serum, lung and kidney. The expression of AQP2 in rat renal tissue was determined with real-time fluorescence quantitative PCR(RT-PCR).RESULTS:(a) In comparison with the normal control, It was found that enforced vital capacity(FVC),1-second forced expiratory volume/forced vital capacity%(FEV1/FVC%), 24 h urine volume content of NO and PGE2 were decreased, while AQP2 m RNA level and content of ADH, Agn Ⅱ, ANP and ET-1were increased in the model group with statistical significance(P < 0.05).(b) In comparison with the model group, It was found that FVC, FEV1, FEV1/FVC%, 24 h urine volume, content of PGE2 and NO decreased, while AQP2 m RNA level, content of ANP,ADH and AngⅡ decreased in the SAT group with statistical significance(P < 0.05).CONCLUSION: SAT might effectively regulate the urine volume in the modeled rats; ADH, Ang Ⅱ,ANP, ET-1, NO and PGE2 might play an important role in the regulation on urine volume by lungs.This might be the mechanisms underpinning the function of lung governing water passage in terms of the theory of Traditional Chinese Medicine.

理中丸对脾虚型腹泻大鼠结肠黏膜屏障IL-22-MUC2/claudin-2信号通路的影响

目的 探讨理中丸对脾虚型腹泻大鼠结肠黏膜IL-22-MUC2/claudin-2信号通路的调控作用。方法 采用番泻叶水煎液1.2 g/mL联合疲劳游泳建立脾虚型腹泻大鼠模型。模型大鼠随机分为模型组、蒙脱石散组(0.9 g/kg)和理中丸低、高剂量组(0.81和3.24 g/kg),每组8只;另选8只正常大鼠作为空白组。连续给药14 d,造模结束后可见大鼠体重减轻,精神倦怠、眯眼聚堆、少动,大便质地偏软或者便溏,肛周污秽,提示模型复制成功。采用比色法检测血清淀粉酶和D-木糖水平,阿利新蓝和过碘酸雪夫(AB-PAS)染色检测结肠黏膜形态结构,蛋白免疫印迹法(Western Blot)检测黏蛋白2(MUC2)和水通道蛋白3/4(AQP3、AQP4)的表达,免疫荧光法检测结肠组织claudin-2蛋白表达,实时荧光定量-PCR(RT-PCR)法检测白介素-22(IL-22)和肠黏膜上皮跨膜蛋白2(claudin-2)基因相对表达量(2-ΔΔCt值)。结果 与空白组比较,模型组大鼠稀便率、稀便级和腹泻指数显著增加,结肠组织黏膜层上皮细胞出现不同程度的萎缩,黏膜表面结构疏松水肿,杯状细胞减少,血清淀粉酶和D-木糖含量减少,结肠组织中MUC2、AQP3和AQP4蛋白表达量相对降低,IL-22 mRNA表达量相对减少和claudin-2 mRNA表达量相对增加,claudin-2蛋白荧光强度明显增强(P<0.05或P<0.01);与模型组比较,理中丸高剂量组大鼠稀便率、稀便级和腹泻指数均明显减少,结肠黏膜病理损伤明显减轻,血清淀粉酶和D-木糖含量增加,结肠组织中MUC2、AQP3和AQP4蛋白表达量相对升高,IL-22 mRNA表达量相对增加,claudin-2 mRNA和claudin-2蛋白表达量相对减少,差异具有统计学意义(P<0.05或P<0.01)。结论 理中丸明显调节脾虚型腹泻大鼠结肠黏膜屏障结构功能,其机制可能与增加结肠AQP3/4表达,调控IL-22-MUC2/claudin-2信号通路有关。

络风宁2号方对慢性心力衰竭利尿剂抵抗患者的临床研究

目的:观察络风宁2号方对慢性心力衰竭利尿剂抵抗患者的心功能及尿液水通道蛋白2(AQP2)表达的影响。方法:选取2019年1月至2021年1月北京中医医院顺义医院收治的慢性心力衰竭合并利尿剂抵抗患者70例作为研究对象,按照随机数字表法分为对照组和观察组,每组35例。将24 h尿量、血浆氨基末端脑钠肽前体(NT-proBNP)、尿液水通道蛋白2(AQP2)水平以及心力衰竭患者的疗效评价标准作为评价指标,分别于治疗前、治疗2周后进行评估。结果:治疗前2组患者24 h尿量、血浆NT-proBNP、尿液AQP2水平比较,差异无统计学意义(P>0.05)。治疗2周后,与治疗前比较,2组患者24 h尿量、血浆NT-proBNP、尿液AQP2水平均有所改善,差异有统计学意义(P<0.01或P<0.05);治疗后观察组24 h尿量明显高于对照组(P<0.05),NT-proBNP水平、尿液AQP2水平均较对照组下降(P<0.05)。2组总有效率比较,差异无统计学意义(P>0.05),但体现出观察组较优的趋势。结论:络风宁2号方能够增加慢性心力衰竭患者的尿量、减轻水钠潴留状态、改善利尿剂抵抗情况,改善心功能;同时本研究也表明联合监测血浆NT-proBNP及尿液AQP2水平,可作为慢性心力衰竭患者水液失衡的新靶点。

恩格列净对慢性心力衰竭大鼠肾脏功能和肾脏AQP2表达的影响及机制研究

目的探究分析恩格列净对慢性心力衰竭(chronic heart failure,CHF)大鼠肾功能和肾脏水通道蛋白2(aquaporin 2,AQP2)表达的影响及可能的作用机制。方法成年雄性SD大鼠采用随机数字表法分为假手术组(Sham组)、慢性心力衰竭组(CHF组)、恩格列净组(Emp组),采用左冠状动脉前降支(left anterior descending coronary artery,LADCA)结扎法构建CHF模型。建模成功后,Emp组给予5 mg/kg恩格列净灌胃,1次/d,Sham组及CHF组给予等体积0.9%生理盐水灌胃,干预4周。观察干预前后各组大鼠精神状态、体质量、毛发、活动、喘鸣音等一般情况变化。苏木精-伊红(hematoxylin-eosin,HE)染色及Masson染色观察肾组织病理变化。全自动生化分析仪检测尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatine,Scr)及血清精氨酸加压素(arginine vasopressin,AVP)含量;实时荧光逆转录定量聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)检测肾组织中AQP2 mRNA含量;Western blot和免疫组织化学检测肾组织中AQP2蛋白表达;Western blot法检测肾脏组织中p-NF-κB p65、p-IκBα蛋白含量。结果Sham组大鼠精神状态好,毛发光泽度好,饮食正常,无喘咳,肾脏组织结果正常,无胶原沉积表现;CHF组大鼠精神状态不佳,毛发暗淡脱发,饮食减少,喘咳明显,肾组织结构紊乱,肾小管扩张,肾间质炎性细胞浸润,肾小球基底膜及肾间质胶原沉积明显;Emp组大鼠精神状态较为正常,毛发光泽度改善,饮食增加,喘咳减少,体质量增加,肾组织结构较为完整,肾小管扩张减轻,肾间质炎性细胞浸润减少,肾间质及肾小球基底膜胶原沉积明显减少。与Sham组比较,CHF组大鼠血清BUN、Scr、AVP等肾功能生化指标水平均明显较高,AQP2 mRNA及蛋白相对表达量均明显较高(P均<0.01);p-NF-κB p65蛋白相对表达量明显较高,p-IκBα蛋白相对表达量则明显较低(P均<0.01)。与CHF组比较,Emp组大鼠血清BUN、Scr、AVP水平均明显降低,AQP2 mRNA及蛋白相对表达量明显较低(P均<0.01);p-NF-κB p65蛋白相对表达量明显较低,p-IκBα蛋白相对表达量则明显较高(P<0.01)。结论恩格列净可通过抑制NF-κB信号通路作用,下调CHF大鼠肾组织AQP2表达,减少肾脏间质间胶原沉积,改善大鼠肾功能。

Aquaporin,beyond a transporter

Aquaporins(AQPs)are one of the most ancient superfamily proteins,which are essential for maintaining the fluid homeostasis of most organisms against various environments.Here,the latest findings for function of AQPs in cell signal transduction in plants are summarized.We also put forward several issues that still need to be addressed in the future.

温肾消水膏皮肤给药舒缓H22肝癌腹水瘤模型小鼠癌性腹水的作用机制研究

目的观察温肾消水膏皮肤给药舒缓H22肝癌腹水瘤模型小鼠癌性腹水的作用机制。方法将9只C57BL/6小鼠随机分为模型组、温肾消水膏组、空白组,每组3只,其中模型组、温肾消水膏组采用腹腔接种H22肝癌细胞悬液的方法建立H22肝癌腹水瘤小鼠模型。空白组和模型组不予药物,温肾消水膏组从接瘤后24 h开始,每日予温肾消水膏外敷腹部,每天4 h,连续10天。于第11天以颈椎脱臼法处死小鼠,取腹水、肾脏组织;用注射器计量腹水量,采用显微镜计数法计算肿瘤细胞数,用蛋白质免疫印迹(Western Blot)法检测肾脏组织的水通道蛋白1(AQP1)、水通道蛋白2(AQP2)、血管内皮生长因子A(VEGF-A)和肿瘤坏死因子α(TNF-α)蛋白表达,用逆转录聚合酶链式反应(RT-PCR)法检测肾脏组织中AQP1、AQP2、TNF-α、VEGF-A mRNA表达水平;对肾脏组织进行苏木素-伊红(HE)染色和马松(Masson)染色,观察肾脏的病理形态学改变。结果与模型组比较,温肾消水膏组腹水量减少,但比较差异无统计学意义(P>0.05)。与模型组比较,温肾消水膏组腹水瘤细胞计数减少,但比较差异无统计学意义(P>0.05)。与空白组比较,模型组、温肾消水膏组小鼠肾脏组织VEGF-A蛋白表达均降低(P<0.05),AQP1、AQP2、TNF-α蛋白表达比较差异无统计学意义(P>0.05)。与模型组比较,温肾消水膏组小鼠肾脏组织AQP1蛋白表达升高(P<0.05),AQP2、TNF-α、VEGF-A蛋白表达比较差异无统计学意义(P>0.05)。与空白组比较,模型组、温肾消水膏组肾脏VEGF-A mRNA表达均降低(P<0.05),AQP1、AQP2、TNF-αmRNA表达比较差异无统计学意义(P>0.05)。与模型组比较,温肾消水膏组小鼠肾脏组织AQP1、AQP2、TNF-α、VEGF-A mRNA表达比较差异无统计学意义(P>0.05)。HE染色结果显示,与空白组比较,温肾消水膏组、模型组小鼠肾小球系膜区增宽,系膜细胞增生,肾小管变性,部分区域肾小球萎缩,肾间质有炎性细胞浸润;与模型组比较,温肾消水膏组小鼠肾间质炎性细胞浸润情况较少,系膜细胞增生减少。Masson染色结果显示,与空白组比较,模型组、温肾消水膏组有大量胶原纤维沉积于肾小球系膜区;与模型组比较,温肾消水膏组胶原纤维较少。结论温肾消水膏有一定的舒缓肝癌癌性腹水作用,其机制可能涉及以下方面:一是温肾消水膏减轻了肾间质炎症,减少肾小球系膜细胞增生,改善了肾脏病理;二是可能与温肾消水膏上调肾脏AQP1、AQP2蛋白表达,调节肾脏VEGF蛋白表达使之接近正常的作用有关。

水通道蛋白2在胆总管结扎大鼠肝硬化肾组织中的表达及丹参注射液对其表达的影响

目的:探讨胆总管结扎(bile duct ligation,BDL)大鼠肝硬化不同阶段肾组织中水通道蛋白2(aquaporins-2,AQP2)表达的变化;用丹参注射液干预肝硬化腹水大鼠,观察丹参注射液对其表达的影响.方法:将70只♂SD大鼠随机分成假手术组、模型组.模型组分别于结扎后1、2、3、4wk麻醉10只动物.假手术组与4wk模型组同批麻醉.4wk末时,再从剩余存活造模大鼠中,随机选取16只腹腔穿刺证实有腹水的大鼠,随机分为治疗组和对照组.从造模第5周开始进行治疗观察.治疗组给予丹参注射液2mL腹腔注射,1次/d,连用2wk.对照组给予蒸馏水2mL腹腔注射,1次/d,连用2wk,6wk末2组同批麻醉.采用HE染色、Masson三色染色确定模型建立情况;肾脏组织标本采用HE染色,光镜下观察组织学变化,应用免疫组织化学方法测定AQP2的表达及分布.结果:造模1-4wk大鼠肾组织AQP2的阳性染色平均吸光度A值(皮质:0.4703±0.0313,0.4832±0.0212,0.5081±0.0417,0.6802±0.0531;髓质:0.4320±0.0237,0.4724±0.0284,0.4796±0.0451,0.5187±0.0612)均高于假手术组(皮质:0.4197±0.0295;髓质:0.4139±0.0152,P<0.05),提示肝硬化进程中,AQP2在肾皮质和肾髓质的表达均逐渐增强.治疗组(皮质:0.4233±0.0521,髓质:0.4158±0.0413)大鼠肾组织AQP2的阳性染色平均A值显著低于对照组(皮质:0.5536±0.0476,髓质:0.4764±0.0536,P<0.05).结论:以BDL法制备的大鼠肝硬化模型中存在着肾脏AQP2表达的上调,提示在肝硬化进程中,肾脏集合管可能参与了水重吸收的增加;而丹参注射液可以下调肝硬化腹水大鼠肾脏AQP2的高表达.

尿液中水孔蛋白-2的检测及其与肾组织中表达的相关性

目的：研究尿液 Ａ Ｑ Ｐ２ 水孔蛋白检测的方法及其与肾脏 Ａ Ｑ Ｐ２ 水孔蛋白表达量之间的相关性，为应用尿液 Ａ Ｑ Ｐ２ 蛋白监测机体的水重吸收状况提供理论依据。 方法：用 Ｗｅｓｔｅｒｎ 印迹分析法测定自由进水和禁水２４ｈ 大鼠肾脏 Ａ Ｑ Ｐ２ 的表达量及尿液 Ａ Ｑ Ｐ２ 的含量，并分析两者之间的相关性。 结果： Ａ Ｑ Ｐ２ 蛋白可在尿液中检测到。尿液 Ａ Ｑ Ｐ２ 与肾脏 Ａ Ｑ Ｐ２ 呈正相关（ ｒ ＝ ０７９９） 。 结论：尿液 Ａ Ｑ Ｐ２ 水平可以反映肾脏集合管 Ａ Ｑ Ｐ２ 蛋白的表达量，是较好的监测机体水重吸收状况的方法。