脓毒症大鼠肾脏AQPs动态变化与水代谢的关系

目的：探讨肾脏AQPs的表达与肾损伤水代谢紊乱的关系。方法：Wistar大鼠采用盲肠结扎穿孔法（CLP）建立脓毒症大鼠模型。观测尿量、尿渗透压和肾功能。免疫组化方法检测AQP1、AQP2蛋白水平,实时荧光定量PCR方法检测AQP1mRNA、AQP2mRNA水平。结果：手术组大鼠肾脏AQP1蛋白及AQP1mRNA表达随着CPL术后时间的延长呈下降趋势,6h开始下降,24h达最低;AQP2蛋白及AQP2-mRNA表达均在术后3h增高、12h、24h明显降低,较假手术组差异有统计学意义（P〈0.05）。大鼠肾脏AQP1、AQP2分别与尿量呈负相关（P〈0.05）,与尿渗透压呈正相关（P〈0.05）。结论：AQP1和AQP2基因通过调节AQP1、AQP2蛋白的表达,参与肾脏水代谢的调节;AQP1、AQP2蛋白及基因的表达异常是导致脓毒症肾损伤水代谢紊乱的重要原因。     

泽黄颗粒对糖尿病大鼠肾脏和尿液AQP2表达的调节效应

目的：探讨肾脏和尿液AQP2的表达与糖尿病肾病（DN）大鼠水液代谢异常关系及泽黄颗粒防治DN效应机制。方法：用高脂高糖饲料加小剂量STZ诱导DM大鼠模型,随机分为糖尿病模型组（DM组）、泽黄颗粒治疗组（Z组）和格列吡嗪对照组（G组）,正常对照组（N组）。于实验第12周末检测BG、HbA1c、Ccr、UAER及血液流变学指标;观察肾组织病理改变;运用Western-Blot、Real-Time PCR法检测肾脏AQP2蛋白及mRNA表达变化,并检测尿液内AQP2浓度的变化。结果：与DM组相比,Z组大鼠BG、HbA1c、UAER、全血黏度、血浆黏度、肾脏AQP2蛋白、AQP2 mRNA表达和尿液内AQP2浓度均明显下降（P〈0.05）,Ccr明显升高（P〈0.01）。与G组相比,Z组大鼠全血黏度、TG、TC水平明显降低（P〈0.05）。结论：泽黄颗粒可下调糖尿病大鼠肾脏和尿液AQP2的表达,这可能是其调节水液代谢的机制和防治DN的基础。     

糖尿病肾病大鼠肾组织α-actinin-4表达及通络泄浊方的干预作用

目的：探讨α-actinin-4在糖尿病肾病大鼠肾组织中表达的变化,并观察通络泄浊方对其表达的影响。方法：采用链脲佐菌素（STZ）法制作大鼠模型,随机分为正常对照组、模型组、通络泄浊方组、科素亚组和通络泄浊方＋科素亚组。采用Realtime-PCR及Westernblot方法检测肾组织α-actinin-4mRNA和蛋白的表达。结果：与正常对照组相比,模型组mRNA表达和蛋白明显降低（P〈0.01）,给药组与模型组相比,mRNA和蛋白表达明显增高（P〈0.01）。结论：α-acti-nin-4的表达降低参与了糖尿病肾病大鼠蛋白尿的发生;通络泄浊方可以通过上调DN大鼠肾组织α-actinin-4mRNA和蛋白的表达而改善大鼠的肾功能及蛋白尿水平。     

血管内皮生长因子和内抑素在STZ大鼠肾脏中的表达及意义

目的：研究血管内皮生长因子（VEGF）和内抑素（ENS）在2型糖尿病大鼠肾脏中的表达状况、比值变化及与肾脏微血管病变的关系。方法：将Wister大鼠设正常对照组和模型组，使用STZ诱导形成2型糖尿大鼠肾病模型，分别于2、4、8、12周，采用RT-PCR的方法观察大鼠肾脏VEGF和ENSmRNA的表达，并观察其比值变化，同时采用免疫组化观察VEGF蛋白的表达变化。结果：（1）糖尿病组2周时肾脏中VEGFmRNA开始上调（P〈0．05），4、8、12周时较正常对照组明显上调（P〈0．01）。（2）糖尿病组2周时肾脏中ENSmRNA的表达开始上调（P〈0，05），8、12周时表达明显上调（P〈0．01）。（3）糖尿病组2周时肾脏的VEGFmRNA／ENSmRNA值未见变化（P〉0．05），4周时升高（P〈0．05），12周时明显升高（P〈0．01）。（4）免疫组化显示糖尿病肾病组2周时VEGF升高，12周时升高更明显。结论：VEGF和ENS同时参与了糖尿病肾病的血管生成调控，两者表达水平的失衡是其新生血管形成的关键。     

水通道蛋白3、4、8在大鼠慢传输型便秘模型结肠黏膜中的表达

目的探讨水通道蛋白（AQP）3、4、8在大鼠慢传输型便秘（STC）模型中的表达情况。方法利用复方地芬诺酯灌胃的方法建立大鼠STC模型,采用RT—PCR方法测定STC组（16只）及对照组大鼠（16只）升、降结肠黏膜的AQP3、4、8mRNA表达情况。结果STC组与对照组升结肠、降结肠AQP3平均相对表达量分别为0．344和0．602（P〈0．05）、0．419和0．509（P〉0．05）：STC组和对照组升结肠、降结肠AQP4平均相对表达量分别为0．764和0．759（P〉0．05）、0．776和0．736（P〉0．05）;AQP8在实验组和对照组升降结肠中未见明显表达。结论AQP3在STC大鼠的升结肠黏膜中表达下调,对水吸收起调节作用：AQP4和AQP8的表达无明显变化。     

LDLr表达失衡在阿霉素肾病大鼠肾小球硬化中的作用

目的：观察阿霉素肾病大鼠肾小球硬化过程中低密度脂蛋白受体（ LDLr）的表达情况,及辛伐他汀对LDLr表达的影响,探讨LDLr在脂质导致肾病大鼠肾脏损害过程中的作用及他汀类药物肾脏保护的机制。方法：雄性SD大鼠随机分为正常对照组、阿霉素肾病组（模型组）和阿霉素肾病辛伐他汀治疗组（治疗组）,12周后收集标本,光镜观察肾小球硬化情况,酶比色法和油红染色法检测肾组织内胆固醇含量,RT-PCR和Western-blot技术分别检测肾组织LDLr、胆固醇调节元件结合蛋白-2（SREBP-2） mRNA和蛋白质表达。结果：模型组出现明显肾小球硬化（P〈0.01）,LDLr、SREBP-2 mRNA和蛋白表达明显增强（P均〈0.01）,肾组织内胆固醇含量明显升高（P〈0.01）；线性回归分析显示肾组织LDLr、SREBP-2蛋白表达上调与肾组织胆固醇酯（CE）含量呈显著正相关性（P均〈0.01）。治疗组肾小球硬化明显减轻（P〈0.01）,LDLr、SREBP-2 mRNA和蛋白表达、肾组织内胆固醇含量均较模型组低（分别P〈0.05或P〈0.01）。结论：肾病大鼠肾小球硬化过程中,肾组织内SREBP-2、LDLr表达明显上调,脂质可能通过过度表达的LDLr途径在肾组织内沉积,加重肾小球硬化；辛伐他汀可能通过下调LDLr途径,减少脂质在肾脏组织内沉积,发挥肾脏保护作用。     

胆道梗阻解除术后肾水通道蛋白2表达变化的实验研究

目的明确水通道蛋白2（aquaporin 2 protein,AQP2）在梗阻性黄疸梗阻解除术后大鼠肾脏的表达情况。方法采用蛋白质印迹（western blotting）法及RT-PCR方法检测在梗阻性黄疸梗阻解除后不同时间的大鼠肾脏AQP2及其mRNA的水平。结果AQP2蛋白水平在0h,24h,72h随时间推移逐渐降低,1周时明显高于解除梗阻0h的水平．但所有实验组均较正常对照组（假手术组）低（P〈0．05）。而AQP2的mRNA水平在梗阻性黄疸梗阻解除后0h．24h,72h随时间推移逐渐性升高,1周时回落,但比0h水平还要高,然而所有实验组均较正常对照组（假手术组）高（P〈0．05）。结论梗阻性黄疸梗阻解除术后肾功能在早期受损逐渐加重,AQP2蛋白水平下调．而AQP2基因水平上调。1周时这些结果仍未恢复正常。AQP2在梗阻性黄疸梗阻解除术后肾功能的变化上可能起到了重要的作用。     

重症急性胰腺炎大鼠p38丝裂原活化蛋白激酶的表达与肺毛细血管内皮屏障损伤

目的研究p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38MAPK）在重症急性胰腺炎（severe acute pancreatitis,SAP）大鼠肺组织中的表达情况,并初步探讨p38MAPK与SAP肺毛细血管内皮屏障损伤的关系。方法将40只健康雄性SD大鼠随机（随机数字表法）分为假手术（SO）组和SAP组,SAP组又再分为3、6、12及24 h 4个时间点组,共5组,每组8只。采用胰胆管逆行注射5%牛磺胆酸钠的方法建立SAP大鼠模型。采用HE染色方法观察大鼠肺和胰腺组织的病理学改变;采用ELISA法检测血清肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）水平;采用免疫组化染色方法检测肺组织中磷酸化p38（p-p38）蛋白和水通道蛋白1（AQP1）的表达水平;采用实时定量荧光PCR法（real-time PCR）检测肺组织中AQP1 mRNA的表达水平。结果 SAP各时间点组大鼠肺组织充血水肿,炎症细胞浸润;胰腺组织可见大片坏死,部分腺叶结构模糊甚至消失;血清TNF-α和IL-1β水平均较SO组高（P〈0.05）。SO组大鼠肺组织中p-p38蛋白仅有微量表达,而在SAP3 h组,p-p38蛋白的表达就明显上调,在6 h时达高峰,24 h时仍高于SO组（P〈0.05）。SAP各时间点组大鼠肺组织中AQP1 mRNA及其蛋白的表达水平均较SO组下降（P〈0.05）,并随着时间的推移而逐渐下降;SAP组大鼠AQP1 mRNA的表达与TNF-α、IL-1β及p-p38蛋白之间均存在负相关关系（r=-0.87,P〈0.05;r=-0.88,P〈0.05;r=-0.78,P〈0.05）。结论肺组织中AQP1蛋白表达的下调是SAP肺毛细血管内皮屏障损伤的重要原因之一,其可能与p38MAPK的激活及炎症因子的过度释放有关。     

益肾降浊冲剂对脾肾气虚型糖尿病肾病Ⅲ期患者尿微量白蛋白的影响

目的：观察益肾降浊冲剂对脾肾气虚型糖尿病肾病（diabetic kidney disease,DKD）Ⅲ期患者的临床疗效及对尿微量白蛋白的影响,探讨益肾降浊冲剂在治疗糖尿病肾病中的作用机制。方法：根据丹麦学者Mogensen糖尿病肾病诊断和分期标准,选取DKDⅢ期患者80例。采用随机对照方法,将80例患者随机分为两组,对照组（40例）单纯用现代医学常规疗法治疗;治疗组（40例）在现代医学常规治疗基础上给予口服益肾降浊冲剂每次10g,1天3次。两组均以2个月为1疗程,观察两组患者治疗前后中医临床症状积分、肝功能、肾功能、尿微量白蛋白水平变化。结果：对照组有2例、治疗组有3例患者因失访未完成观察。两组患者治疗后肾功能较治疗前均未见明显改善（P〉0.05）;治疗组患者中医症状积分较治疗组改善更为明显（P〈0.05）;治疗组与对照组相比尿微量白蛋白下降更明显（P〈0.05）;两组患者治疗期间均未见明显副作用。结论：益肾降浊冲剂加常规西药治疗能降低脾肾气虚型DKDⅢ期患者尿微量白蛋水平,改善临床症状。     

连黄降浊颗粒对5/6肾切除大鼠肾功能和肾皮质细胞外基质积聚的影响

目的：观察连黄降浊颗粒对5/6肾切除大鼠肾功能和肾皮质细胞外基质（ECM）积聚的影响。方法：将45只Wistar大鼠随机取8只为正常对照组,余用5/6肾切除法建立慢性肾衰竭（CRF）动物模型,将造模成功后的大鼠随机分为模型组、尿毒清颗粒组（对照组）、连黄降浊颗粒低剂量组（低剂量组）、连黄降浊颗粒高剂量组（高剂量组）,分别给予相应浓度和剂量的药物。实验期间测定大鼠的肾功能、尿蛋白,治疗12周后观察其肾脏病理改变,免疫组化法检测肾小球Ⅳ型胶原（ColⅣ）、纤连蛋白（FN）和肾皮质基质金属蛋白（MMP-2）和金属蛋白酶组织抑制物（TI MP-2）蛋白表达。结果：连黄降浊颗粒能明显改善CRF大鼠肾功能、减少尿蛋白的排出（P〈0.05～0.01）,减轻肾脏病理损害;连黄降浊颗粒能下调TI MP-2蛋白表达,增加MMP-2活性,抑制了FN、ColⅣ在肾组织的表达（P〈0.01）,减轻ECM积聚。结论：连黄降浊颗粒可明显改善肾功能,增加肾脏MMP-2活性,降低TI MP-2表达,增加ECM降解,减轻肾小球硬化。     

Regulation of aquaporins and sodium transporter proteins in the solitary kidney in response to partial ureteral obstruction in neonatal rats

Unilateral ureteral obstruction (UUO) impairs function of the obstructed kidney, and the contralateral nonobstructed kidney compensates depending on the degree and duration of UUO. This study aimed to determine the hemodynamic and molecular changes in the solitary kidney in response to partial ureteral obstruction (PUO) where any compensation from the contralateral kidney was eliminated so that all observed changes in the kidney tissue occurred in the kidney with PUO. Newborn rats were subjected to unilateral left nephrectomy (UNX) within the first 48 h of life and a subset of UNX rats was subjected to severe PUO of the right kidney at day 14. Renal blood flow and whole kidney volume were measured with MRI at week 10. The renal protein abundance of aquaporin 1 (AQP1), AQP2 and AQP3 as well as Na,K-ATPase, NaPi-2 (type 2 sodium-phosphate cotransporter) and NHE3 (type 3 sodium-proton exchanger) were examined by immunoblotting and immunocytochemistry. At 10 weeks of age, the protein abundance of AQP2, AQP3, Na,K-ATPase, NaPi-2 and NHE3 were increased in response to PUO. In contrast, AQP1 expression was markedly decreased compared to sham-operated rats. These findings were confirmed by immunohistochemistry. GFR, urine osmolality and urine sodium excretion were reduced and kidney weight increased in response to PUO. In conclusion, the present study demonstrated major changes in the protein abundance of renal AQP1, AQP2 and AQP3 and sodium transporters in the solitary PUO kidney. These changes were paralleled by decreased urinary sodium excretion and a significant reduction in urinary osmolality from the obstructed kidney, suggesting a functional association between the molecular changes and the ability of the obstructed kidney to handle sodium and water in this solitary kidney model.     

猪苓汤对阿霉素肾病大鼠肾脏AQP2表达的影响

目的:探讨猪苓汤对阿霉素肾病大鼠水通道蛋白2表达的影响。方法:110只SPF级雄性SD大鼠随机分为空白组、假手术组、模型组,尾静脉注射阿霉素14 d后,24 h尿蛋白定量≥100 mg提示模型复制成功,将模型大鼠再次随机分为模型组、猪苓汤组、呋塞米组,分别于灌胃2、4、6、8周处死部分大鼠,留取血液、尿液、肾脏标本,半自动生化仪检测尿蛋白含量、肾功能等生化指标,HE染色观察肾脏病理改变,RT-PCR法检测AQP2 mRNA表达,Western Blot法检测AQP2蛋白表达。结果:(1)生化指标:灌胃8周,猪苓汤组平均尿量高于模型组(P<0.05),其他生化指标比较,各造模组之间差异无统计学意义;(2)AQP2 mRNA:注射后14 d,模型组平均表达量高于空白组,差异具有统计学意义(P<0.05),猪苓汤组在灌胃2、4、6、8周时AQP2 mRNA平均表达量均低于模型组(P<0.05);(3)AQP2蛋白:注射后14 d,模型组平均表达量高于空白组,差异具有统计学意义(P<0.05),猪苓汤组在灌胃4、6、8周时AQP2蛋白平均表达量均低于模型组(P<0.05)。结论:猪苓汤的利尿作用可能通过下调AQP2 mRNA和蛋白表达而实现。     

Reduced renal expression of AQP2, p-AQP2 and AQP3 in haemorrhagic shock-induced acute renal failure.

BACKGROUND: The aims of this study were to investigate the changes in the expression levels of renal aquaporins (AQPs) in response to haemorrhagic shock (HS) in rats and whether a change in the expression of AQPs was associated with parallel changes in urinary concentration. METHODS: HS was induced by withdrawal of blood through the femoral artery in rats. A mean arterial blood pressure (MAP) of 40 mmHg was maintained for 1 h before blood was reinfused, and rats were kept in metabolic cages for urine measurements. Two days after HS, we examined the abundance of AQPs in kidney by semiquantitative immunoblotting. RESULTS: HS rats (n = 13) developed acute renal insufficiency (creatinine clearance was 5.5 +/- 0.4 vs 6.9 +/- 0.3 ml/min/kg in sham-operated rats, n = 13, P < 0.05) and decreased urine osmolality (888 +/- 88 vs 1799 +/- 110 mosmol/kg H(2)O, P < 0.05). Consistent with this, semiquantitative immunoblotting revealed that the abundance of AQP2, phosphorylated (Ser256) AQP2 (p-AQP2) and AQP3 in whole kidney was significantly decreased after 2 days to 33 +/- 4, 41 +/- 9 and 35 +/- 14% of sham levels, respectively (P < 0.05). Also, the abundance of AQP2, p-AQP2 and AQP3 in inner medulla was markedly decreased to 36 +/- 8, 39 +/- 10 and 34 +/- 16% of sham levels (P < 0.05). In contrast, the abundance of AQP1 was not significantly changed compared with sham levels. CONCLUSIONS: The expression of the collecting duct water channel AQP2, p-AQP2 and AQP3 was significantly downregulated after HS, which may play an important role in the impaired urinary concentrating ability in HS-induced acute renal failure.     

阻塞性黄疸时L-精氨酸对肾功能的保护作用

目的：研究阻塞性黄疸（OJ）时，L－精氨酸（L－Arg)对肾功能的保护作用。方法：胆总管结扎大鼠30只，随机分成生理盐水对照（NS）组、L－精氨酸（L－Arg)组和L－硝基精氨酸（L－NNA）组，每组10只。胆总管结扎后第2天起分别腹腔注射1ml NS、1ml L-Arg(500mg/kg)、1ml L-NNA(10mg/kg)，连用9d；假手术（SO）组用1ml NS腹腔注射。观察各组肾功能的变化，同时测定血和肾组织内皮素（ET）、一氧化氮（NO）水平、一氧化氮合酶（NOS）活性和丙二醛（MDA）的含量。并用图像分析检测ET1 mRNA和NOS mRNA表达的部位及量的变化。结果：用L－Arg后，血和肾组织NOS活性增加，肾组织ET1 mRNA表达减少，血和肾组织ET下降，NO升高；同时伴有内生肌酐清除率（Ccr)、肾皮质平均血流（RCBF）的升高，肾组织MDA含量降低。结论：L－Arg通过增强血和肾组织NOS活性来增加体内NO水平、抑制ET1 mRNA表达、降低体内ET水平，从而提高Ccr与RCBF，减轻阻塞性黄疸时的肾功能损伤。     

充血性心力衰竭时肾脏水通道蛋白mRNA表达的改变及意义

目的 研究在不同程度的充血性心功能衰竭（心衰）时肾脏水通道蛋白2（AQP2），上皮性钠通道（ENaC)和髓襻升支粗段的Na-K-2Cl转运子（rBSC1)表达的情况。方法 将SD大鼠通过腹主动脉－下腔静脉穿刺造瘘和冠状动脉结扎的方法建成不同的心衰模型，设正常对照组，穿刺造瘘1孔组，穿刺造瘘3孔组和冠脉结扎组。用Doppler超声心动图及心脏秤重的方法比较其心功能的各项参数，并用RT－PCR的方法检测肾脏AQP，ENaCα亚单位和rBSC1mRNA表达。结果 穿刺造瘘大鼠心功能损害较轻，而冠脉结扎大鼠心功能严重失代偿。AQP2仅在冠脉结扎大鼠肾皮质表达增高，而rBSC1在造瘘1孔，3孔和结扎大鼠的肾髓质表达均显著上调。肾皮质αENaC含量3组心衰大鼠都显著增高，而在肾髓质仅冠脉结扎大鼠明显升高，造瘘大鼠虽有上升趋势，但差异无显著性意义。结论 在不同类型的充血性心力衰竭大鼠模型中存在着肾脏AQP，rBSC1和ENaC mRNA表达的上调，其中rBSC1增高可能和心脏受累早期肾脏排钠障碍有一定关系。     

Acute Rejection After Rat Renal Transplantation Leads to Downregulation of Na+ and Water Channels in the Collecting Duct

Renal transplantation is associated with alterations of tubular functions and of the renin-angiotensin-aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis-Brown-Norway (LBN) rats received a kidney from LBN-rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT-PCR and Western-blot analysis. Intracellular Na(+) was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na(+)-channel (ENaC) and mRNA and protein expression of the water-channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum- and glucocorticoid-kinase (Sgk1) was decreased and that of the ubiquitin-protein ligase Nedd4-2 was increased. These changes were absent under CsA-therapy and in syngeneic model. Expression and function of the Na(+)-K(+)-ATPase, expression of the secretory K(+)-channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1- and increased Nedd4-2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.     

Functions of aquaporin 1 and α-epithelial Na+ channel in rat acute lung injury induced by acute ischemic kidney injury

Purpose

To establish a rat model of acute ischemic kidney injury by continually occluding the bilateral renal artery and renal veins, the functions of α-epithelial Na⁺ channel (α-ENaC) and aquaporin (AQP1) in lung injury induced by acute kidney injury (AKI) were examined and compared with lung injury induced by endotoxin.

Methods

Male Wistar rats were randomly divided into three groups: control group, AKI group, and sepsis group. The concentrations of AQP1 and α-ENaC in the lung tissue were detected. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the serum and bronchoalveolar lavage fluid were also detected.

Results

The arterial blood pH in AKI group and PaO₂ in sepsis group decreased 2 h after the experiment. A significant pulmonary interstitial and alveolar space edema, which showed a typical pathological change in acute lung injury, was found in AKI and sepsis group 8 h after the experiment. Two hours after the experiment, the concentration of TNF-α and IL-6 in the serum and bronchoalveolar lavage fluid (BALF) in AKI and sepsis group increased, whereas the pulmonary expression of AQP1 and α-ENaC decreased. The pulmonary AQP1 and α-ENaC of the rats were negatively correlated with TNF-α and IL-6 in BALF. The relevance among AQP1, α-ENaC, TNF-α, and IL-6 in sepsis group was higher than that in AKI group.

Conclusion

The TNF-α and IL-6 levels increased significantly and the pulmonary expression of AQP1 and α-ENaC declined at the early stage of AKI.