郭曦; 冯锦锦; 闫少华; 文一博; 文建国

doi:10.3760/cma.j.issn.2095-428X.2019.05.007

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• 泌尿生殖系统疾病 •

促红细胞生成素对双侧输尿管梗阻解除后幼鼠肾脏水通道蛋白-1表达及功能的影响

中华实用儿科临床杂志, 2019,34(5) : 347-351. DOI: 10.3760/cma.j.issn.2095-428X.2019.05.007

摘要

目的

探讨促红细胞生成素(EPO)对双侧输尿管梗阻-梗阻解除(BUO-R)后幼鼠肾脏水通道蛋白-1(AQP-1)表达及功能的影响。

方法

幼年雄性SD大鼠48只，随机号码表法分为双侧输尿管完全梗阻(BUO)组(n＝6)、双侧输尿管梗阻-梗阻解除(BUO-R)组(n＝12)、BUO-R＋EPO组(n＝12)及假手术组(Sham组)(n＝18)。实验组采用输尿管结扎法建立双侧输尿管完全梗阻模型，BUO组梗阻24 h后处死；BUO-R组和BUO-R＋EPO组梗阻24 h后解除梗阻，BUO-R＋EPO组于解除梗阻后1 h、1 d、3 d、5 d给予EPO腹腔注射(500 IU/kg)，BUO-R组相同时间点给予同等剂量的9 g/L盐水，分别在解除梗阻后3 d、7 d处死幼鼠；Sham组仅分离输尿管，不予结扎，分别于梗阻术后24 h、解除梗阻后3 d、7 d处死幼鼠，各组幼鼠处死前取双肾标本并采集血液标本。解除梗阻后将幼鼠放置于代谢笼中，记录24 h饮水量和尿液量，并收集尿液用于检测尿液渗透压的变化。检测各组幼鼠血浆渗透压、血肌酐(Cr)、尿素氮(BUN)水平，采用免疫组织化学、Western blot检测各组肾脏组织中AQP-1蛋白表达水平。

结果

BUO-R后3 d，24 h饮水量和尿液量BUO-R组最多，BUO-R＋EPO组次之，Sham组最少(P<0.05)；尿液渗透压Sham组最高，BUO-R＋EPO组次之，BUO-R组最低(P<0.05)；血浆渗透压、Cr、BUN BUO-R组最高，BUO-R＋EPO组次之，Sham组最低，但均低于BUO组(P<0.05)。梗阻解除后7 d，Sham组无明显变化，BUO-R组和BUO-R＋EPO组以上各项指标逐渐恢复，但仍未达到正常水平(P<0.05)，BUO-R组与BUO-R＋EPO组比较差异有统计学意义(P<0.05)。免疫组织化学结果显示Sham组染色强度最强，BUO-R＋EPO组次之，BUO-R组再次，BUO组最弱；解除梗阻后7 d与解除梗阻后3 d相比，BUO-R＋EPO组和BUO-R组染色强度均增强，仍低于Sham组。经Western blot进一步验证，各组幼鼠肾脏AQP-1表达量Sham组最高，BUO-R＋EPO组次之，BUO-R组再次，BUO组最低(P<0.05)；解除梗阻后7 d与解除梗阻后3 d相比，BUO-R＋EPO组、BUO-R组表达量均增高，但仍低于Sham组(P<0.05)。

结论

EPO能够促进BUO-R幼鼠肾脏AQP-1蛋白表达及功能恢复。

引用本文: 郭曦, 冯锦锦, 闫少华, 等. 促红细胞生成素对双侧输尿管梗阻解除后幼鼠肾脏水通道蛋白-1表达及功能的影响 [J] . 中华实用儿科临床杂志, 2019, 34(5) : 347-351. DOI: 10.3760/cma.j.issn.2095-428X.2019.05.007.

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先天性肾积水(CHN)是常见的小儿泌尿外科疾病，多由先天性解剖学异常而引起。随着积水程度加重，肾功能损害不断进展，最终导致肾衰竭。目前，手术是CHN的主要临床治疗方法。临床发现即使手术解除梗阻，术后一段时间仍有大量稀释尿，易造成水、电解质紊乱^[1]。水通道蛋白-1(AQP-1)在肾脏近曲小管等重吸收过程及尿液浓缩中起重要作用，特别是在髓袢降支细段与逆流倍增机制中发挥关键作用。有研究发现小儿先天性积水肾脏中AQP-1蛋白及其mRNA水平的表达均下调^[2,3]，郑妍和赵琦^[4]发现与健康儿童相比，CHN患儿肾脏AQP-1蛋白及基因表达水平明显下降，与Li等^[3]研究结果相一致。因此推测梗阻解除后一段时间内肾脏AQP-1未恢复原有水平是患者术后仍有大量稀释尿液的原因之一。本研究团队王焱等^[5]发现促红细胞生成素(EPO)可促进输尿管梗阻-解除梗阻(BUO-R)大鼠肾脏AQP-2 mRNA、蛋白表达及功能恢复，但EPO能否促进输尿管BUO-R后AQP-1表达及肾功能恢复，尚未见文献报道。本研究通过对BUO-R幼鼠进行EPO干预，探讨其能否促进BUO-R幼鼠肾脏AQP-1表达及功能的恢复。

1　材料与方法

1.1　实验动物及分组

6～7周幼年雄性SD大鼠48只(河南省实验动物中心，许可证号：SCXK豫20170001)，体质量(160±10) g，根据随机号码表法分为BUO组(n＝6)、BUO-R组(n＝12)、BUO-R＋EPO组(n＝12)及假手术组(Sham组)(n＝18)。大鼠均以啮齿类动物标准饮食，自由饮水，12 h∶12 h自动昼夜循环，温度(21±2) ℃，湿度(55±2)%。

1.2　动物模型制作与标本收集

48只SD大鼠经100 g/L水合氯醛水溶液(腹腔注射，3～5 mL/kg)麻醉后至于保温板上，备皮消毒后经腹正中切口暴露双侧输尿管，钝性分离输尿管，用长4～5 mm的PE50管包裹游离段输尿管，5-0无菌丝线结扎，逐层关闭腹腔，建立双侧输尿管完全梗阻的幼鼠模型。BUO组梗阻24 h后处死，BUO-R组和BUO-R＋EPO组梗阻24 h后拆除PE50管，使输尿管恢复畅通。BUO-R＋EPO组于解除梗阻后1 h、1 d、3 d、5 d给予EPO腹腔注射(500 IU/kg)，分别在解除梗阻后3 d、7 d处死6只幼鼠；BUO-R组相同时间点给予同等剂量的等渗盐水(9 g/L)，之后处理方法同BUO-R＋EPO组；Sham组仅分离输尿管，不予结扎，分别于术后24 h、3 d、7 d各处死6只幼鼠，各组幼鼠处死前取双肾标本并采集血液3 mL标本。幼鼠处死前放置于代谢笼中，记录24 h饮水量和尿液量，并收集尿液用于检测尿液渗透压的变化。右肾置于冻存管中液氮冷冻后，－80 ℃冰箱保存，用于Western blot检测蛋白表达。左肾标本置于40 g/L甲醛溶液中固定24 h，供免疫组织化学检测。血液抽取后，立即以2 054.3×g离心5 min，取上清，－20 ℃冰箱保存，用于检测血浆渗透压、肌酐(Cr)和尿素氮(BUN)水平。

1.3　尿液渗透压测定

采用Oso-M型全自动冰点渗透压仪(德国Gonatec公司生产)测定尿液标本。标准液校准仪器后吸取20 μL尿液标本进行测定，每个标本重复测定3次，结果取其平均值。

1.4　血生化检查

应用全自动生化分析仪测定各组血液标本渗透压、Cr和BUN水平。

1.5　免疫组织化学

40 g/L甲醛溶液固定标本，常规石蜡包埋、切片，按照SABC法免疫组织化学步骤进行操作。一抗采用兔抗鼠AQP-1多克隆抗体(稀释浓度1∶300)，二氨基联苯胺(DAB)显色盒显色，蒸馏水充分冲洗，苏木精复染、盐酸乙醇分化，梯度乙醇脱水，二甲苯透明，树胶封片。

1.6　Western blot

加入蛋白上样缓冲液，100 ℃煮沸10 min。制作十二烷基硫酸钠-聚丙烯酰胺凝胶，蛋白上样，恒压电泳，冰上85 V转膜1 h，封闭液常温封闭2 h，一抗(1∶1 000)孵育，4 ℃过夜，洗膜3次30 min，二抗(1∶2 000)室温孵育1 h，再次洗膜3次30 min，暗室曝光。胶片经凝胶灰度值分析软件分析，以各组AQP-1条带灰度值除以相应内参条带灰度值，比较各组之间的相对值。

1.7　统计学处理

采用SPSS 22.0统计学软件进行数据处理分析，数据采用±s表示。计量资料正态性检验采用Shapiro-Wilk正态性检验，多组间比较采用单因素方差分析，各组间两两比较采用LSD法，P<0.05为差异有统计学意义。

2　结果

2.1　24 h饮水量、尿液量变化

解除梗阻后3 d、7 d，BUO-R组24 h饮水和尿液量最多，BUO-R＋EPO组次之，Sham组最少。与解除梗阻后3 d相比，解除梗阻后7 d BUO-R组和BUO-R＋EPO组的24 h饮水和尿液量减少，BUO-R组显著减少，Sham组无明显变化，见表1。

点击查看表格

表1

各组幼鼠24 h饮水量和尿液量比较(mL，±s)

Table 1

24－hour water intake and urine volume of young rats in each group(mL，±s)

表1

各组幼鼠24 h饮水量和尿液量比较(mL，±s)

Table 1

24－hour water intake and urine volume of young rats in each group(mL，±s)

组别	只数	24 h饮水量		24 h尿液量
组别	只数	解除梗阻后3 d	解除梗阻后7 d	解除梗阻后3 d	解除梗阻后7 d
假手术组	6	19.2±2.5	20.6±3.3	10.1±1.8	10.2±1.7
BUO-R组	6	66.4±5.5^a	41.4±3.4^a	31.2±3.0^a	19.4±1.8^a
BUO-R＋EPO组	6	37.8±2.2^ab	31.3±2.5^ab	18.3±0.8^ab	14.8±2.3^ab
F值		243.510	65.020	158.745	70.054
P值		<0.05	<0.05	<0.05	<0.05

注：BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；^a与Sham组相比，差异有统计学意义(P<0.05)；^b与BUO-R组相比，差异有统计学意义(P<0.05)。在同一组别解除梗阻后3 d与解除梗阻后7 d相比，BUO-R组、BUO-R＋EPO组差异均有统计学意义(均P<0.05)；Sham组差异无统计学意义(P>0.05)　BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；^acompared with Sham group，the difference was statistically significant(P<0.05)；^bcompared with BUO－R group，the difference was statistically significant(P<0.05)．Three days after relief of obstruction comparing with 7 days after relief of obstruction in the same group，there were significant differences in BUO－R group and BUO－R＋EPO group (all P<0.05)，there was no significant difference in Sham group (P>0.05)

2.2　尿液渗透压检测

解除梗阻后3 d、7 d，Sham组尿液渗透压最高，BUO-R＋EPO组次之，BUO-R组最低。与解除梗阻后3 d相比，解除梗阻后7 d BUO-R组和BUO-R＋EPO组尿液渗透压升高，BUO-R＋EPO组尿液渗透压升高更显著，Sham组无明显变化，见表2。

点击查看表格

表2

各组幼鼠尿液渗透压比较(mmol/L，±s)

Table 2

Urine osmotic pressure of young rats in each group(mmol/L，±s)

表2

各组幼鼠尿液渗透压比较(mmol/L，±s)

Table 2

Urine osmotic pressure of young rats in each group(mmol/L，±s)

组别	只数	尿液渗透压
组别	只数	解除梗阻后3 d	解除梗阻后7 d
Sham组	6	1 933±77	1 914±75
BUO-R组	6	649±52^a	812±64^a
BUO-R＋EPO组	6	997±70^ab	1 360±53^ab
F值		584.874	431.916
P值		<0.05	<0.05

注：BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；^a与Sham组相比，差异有统计学意义(P<0.05)；^b与BUO-R组相比，差异有统计学意义(P<0.05)。在同一组别解除梗阻后3 d与解除梗阻后7 d相比，BUO-R组、BUO-R＋EPO组差异均有统计学意义(均P<0.05)；Sham组差异无统计学意义(P>0.05)　BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；^acompared with Sham group，the difference was statistically significant(P<0.05)；^bcompared with BUO－R group，the difference was statistically significant(P<0.05)；3 days after relief of obstruction comparing with 7 days after relief of obstruction in the same group，there were significant differences in BUO－R group and BUO－R＋EPO group (all P<0.05)，there was no significant difference in Sham group (P>0.05)

2.3　血浆渗透压和肾功能变化检测

血浆渗透压：Sham组梗阻24 h、解除梗阻后3 d、7 d血浆渗透压无变化；双侧输尿管梗阻24 h，BUO组血浆渗透压高于Sham组；解除梗阻后3 d，BUO-R组、BUO-R＋EPO组血浆渗透压低于BUO组，高于Sham组，BUO-R组与BUO-R＋EPO组血浆渗透压相比无明显差异；解除梗阻后7 d，与解除梗阻后3 d相比，BUO-R组无明显变化，BUO-R＋EPO组降低，仍高于Sham组；解除梗阻后7 d，BUO-R＋EPO组与BUO-R组相比血浆渗透压降低更显著，见表3。

点击查看表格

表3

各组幼鼠血浆渗透压变化(mmol/L，±s)

Table 3

Plasma osmotic pressure of young rats in each group(mmol/L，±s)

表3

各组幼鼠血浆渗透压变化(mmol/L，±s)

Table 3

Plasma osmotic pressure of young rats in each group(mmol/L，±s)

组别	只数	血浆渗透压
组别	只数	梗阻24 h	解除梗阻后3 d	解除梗阻后7 d
BUO组	6	346±11
假手术组	6	280±6	278±4	280±2
BUO-R组	6		320±3^a	317±5^a
BUO-R＋EPO组	6		315±3^ab	291±4^ab
F值			122.496	127.534
P值			<0.05	<0.05

注：BUO组：双侧输尿管梗阻组；BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；Sham组梗阻24 h、解除梗阻后3 d、解除梗阻后7 d差异无统计学意义(P>0.05)；BUO组与Sham组相比，差异有统计学意义(P<0.05)；^a与Sham组和BUO组相比，差异有统计学意义(P<0.05)；^b与BUO-R组相比，差异有统计学意义(P<0.05)；同一组别解除梗阻后3 d与解除梗阻后7 d相比，BUO-R＋EPO组差异有统计学意义(P<0.05)；BUO-R组差异无统计学意义(P>0.05)　BUO group：bilateral ureter obstruction group；BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；In Sham group，compared with BUO 24 h，3 days after relief of obstruction，7 days after relief of obstruction，there was no significant difference (P>0.05)；BUO group compared with Sham group，the differences were statistically significant(P<0.05)；^acompared with Sham group or BUO group，the differences were statistically significant(all P<0.05)；^bcompared with BUO－R group，the difference was statistically significant(P<0.05)；3 days after relief of obstruction compared with 7 days after relief of obstruction in the same group，there were significant differences in BUO－R ＋ EPO group (P<0.05)；there was no significant difference in BUO－R group(P>0.05)

肾功能：Sham组梗阻24 h、解除梗阻后3 d、7 d血Cr、BUN无变化；双侧输尿管梗阻24 h，BUO组血Cr、BUN显著高于Sham组；解除梗阻后3 d，BUO-R组、BUO-R＋EPO组血Cr、BUN与BUO组相比明显降低，与Sham组相比较高，BUO-R＋EPO组血Cr、BUN低于BUO-R组；解除梗阻后7 d，与解除梗阻后3 d相比，BUO-R组、BUO-R＋EPO组血Cr、BUN均降低，仍比Sham组高；解除梗阻后7 d，BUO-R＋EPO组血Cr、BUN低于BUO-R组，见表4。

点击查看表格

表4

各组幼鼠血肌酐及血尿素氮变化(±s)

Table 4

Serum creatinine and blood urea nitrogen of young rats in each group(±s)

表4

各组幼鼠血肌酐及血尿素氮变化(±s)

Table 4

Serum creatinine and blood urea nitrogen of young rats in each group(±s)

组别	只数	血肌酐(μmol/L)			血尿素氮(mmol/L)
组别	只数	梗阻24 h	解除梗阻后3 d	解除梗阻后7 d	梗阻24 h	解除梗阻后3 d	解除梗阻后7 d
BUO组	6	233.3±3.8			115.6±3.1
假手术组	6	53.7±0.2	53.6±0.7	53.3±0.6	6.0±0.3	6.0±0.5	6.0±0.5
BUO-R组	6		151.1±6.0^a	87.7±2.1^a		56.7±2.8^a	26.8±0.8^a
BUO-R＋EPO组	6		129.6±7.8^ab	83.6±2.1^ab		42.9±4.4^ab	24.1±0.6^ab
F值			793.609	4 792.675		943.496	3 656.460
P值			<0.05	<0.05		<0.05	<0.05

注：BUO组：双侧输尿管梗阻组；BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；Sham组梗阻24 h、解除梗阻后3 d、解除梗阻后7 d差异无统计学意义(P>0.05)；BUO组与Sham组相比，差异有统计学意义(P<0.05)；^a与Sham组和BUO组相比，差异有统计学意义(均P<0.05)；^b与BUO-R组相比，差异有统计学意义(P<0.05)；同一组别解除梗阻后3 d与解除梗阻后7 d相比，BUO-R＋EPO组2项指标差异有统计学意义(P<0.05)；BUO-R组2项指标差异有统计学意义(P<0.05)　BUO group：bilateral ureter obstruction group；BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；in Sham group，compared with BUO 24 h，3 days after relief of obstruction，7 days after relief of obstruction，there was no significant difference (P>0.05)；BUO group compared with Sham group，the difference was statistically significant(P<0.05)；^acompared with Sham group or BUO group，the differences were statistically significant(all P<0.05)；^bcompared with BUO－R group，the difference was statistically significant(P<0.05)；3 days after relief of obstruction compared with 7 days after relief of obstruction in the same group，there were significant differences in two indicators of BUO－R ＋ EPO group(P<0.05) and BUO－R group(P<0.05)

2.4　免疫组织化学测定结果

AQP-1阳性细胞呈棕黄色，主要集中在近曲小管，位于上皮细胞的顶质膜。总体上看，Sham组染色强度最强，BUO-R＋EPO组次之，BUO-R组再次，BUO组最弱，但仍可见微弱表达。Sham组梗阻24 h、解除梗阻后3 d、7 d染色强度无明显变化；解除梗阻后7 d与解除梗阻后3 d相比，BUO-R＋EPO组、BUO-R组染色强度均增强，但仍低于Sham组。组织学上可见BUO组、BUO-R组和BUO-R＋EPO组近曲小管管壁变薄、管腔增大，明显不同于Sham组，见图1。

点击查看大图

图1

各组幼鼠肾脏AQP-1表达的免疫组织化学染色(×400，箭头示近曲小管)

Figure 1

Immunohistochemical map of AQP－1 expression in kidneys of young rats in each group (×400，the red arrow showing proximal convoluted tubules)

点击查看大图

注：BUO组：双侧输尿管梗阻组；BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；R-3 d、7 d：解除梗阻后3 d、7 d　BUO group：bilateral ureter obstruction group；BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；R－3 d，7 d：3 or 7 days after relief of obstruction

图1

各组幼鼠肾脏AQP-1表达的免疫组织化学染色(×400，箭头示近曲小管)

Figure 1

Immunohistochemical map of AQP－1 expression in kidneys of young rats in each group (×400，the red arrow showing proximal convoluted tubules)

2.5　Western blot结果

Sham组相对表达量最高，BUO-R＋EPO组次之，BUO-R组再次，BUO组最低。Sham组梗阻24 h、解除梗阻后3 d、7 d相对无明显变化；解除梗阻后7 d与解除梗阻后3 d相比，BUO-R＋EPO组、BUO-R组表达量均增高，但仍低于Sham组，见表5、图2。Western blot结果与免疫组织化学结果一致。

点击查看大图

图2

各组幼鼠肾脏AQP-1表达的Western blot图

Figure 2

Western blot results of renal AQP－1 expressions in young rats of each group

点击查看大图

注：BUO组：双侧输尿管梗阻组；BUO 24 h：双侧输尿管梗阻24 h；BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；R-3 d、7 d：解除梗阻后3、7 d；AQP-1：水通道蛋白-1　BUO group：bilateral ureter obstruction group；BUO 24 h group：bilateral ureter obstruction 24 h group；BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；R－3 d，7 d：3 or 7 days after relief of obstruction；AQP－1：aquaporin－1

图2

各组幼鼠肾脏AQP-1表达的Western blot图

Figure 2

Western blot results of renal AQP－1 expressions in young rats of each group

点击查看表格

表5

AQP-1在各组幼鼠肾脏的相对表达量(±s)

Table 5

The relative quantitive of renal AQP－1expressions in young rats of each group(±s)

表5

AQP-1在各组幼鼠肾脏的相对表达量(±s)

Table 5

The relative quantitive of renal AQP－1expressions in young rats of each group(±s)

组别	只数	AQP－1相对表达量
组别	只数	梗阻24 h	解除梗阻后3 d	解除梗阻后7 d
BUO组	6	0.22±0.46
假手术组	6	2.32±0.80	2.32±0.90	2.35±0.70
BUO-R组	6		0.52±0.86^a	0.83±0.46^a
BUO-R＋EPO组	6		1.17±0.07^ab	1.97±0.11^ab
F值			261.899	470.618
P值			<0.05	<0.05

注：BUO组：双侧输尿管梗阻组；BUO-R组：双侧输尿管梗阻-解除梗阻组；BUO-R＋EPO组：双侧输尿管梗阻-解除梗阻＋促红细胞生成素干预组；Sham组梗阻24 h、解除梗阻后3 d、解除梗阻后7 d差异无统计学意义(P>0.05)；BUO组与Sham组相比，差异有统计学意义(P<0.05)；^a与Sham组及BUO组相比，差异有统计学意义(P<0.05)；^b与BUO-R组相比，差异有统计学意义(P<0.05)；同一组别解除梗阻后3 d与解除梗阻后7 d相比，BUO-R＋EPO组差异有统计学意义(P<0.05)；BUO-R组差异无统计学意义(P>0.05)　BUO group：bilateral ureter obstruction group；BUO－R group：bilateral ureter obstruction－released group；BUO－R＋EPO group：bilateral ureter obstruction－released＋erythropoietin group；In Sham group，compared with BUO 24 h，3 days after relief of obstruction，7 days after relief of obstruction，there was no significant difference (P>0.05)；BUO group compared with Sham group，the differences were statistically significant(P<0.05)；^acompared with Sham group or BUO group，the difference was statistically significant(P<0.05)；^bcompared with BUO－R group，the difference was statistically significant(P<0.05)；3 days after relief of obstruction comparing with 7 days after relief of obstruction in the same group，there were significant differences in BUO－R＋EPO group (P<0.05)；there was no significant difference in BUO－R group(P>0.05)

3　讨论

BUO是常见的泌尿系统疾病，其最常见的原因是肾盂输尿管连接部梗阻(PUJO)。临床发现即使解除梗阻，患儿术后一段时间仍有大量稀释尿。Li等^[6]建立双侧输尿管梗阻大鼠模型，解除梗阻后对其AQP-1表达进行检测，发现解除梗阻后长时间内AQP-1表达水平下调，30 d后AQP-1表达虽有所回升但仍低于正常水平。AQP-1主要表达在肾单位的近曲小管和髓襻降支细段上皮细胞的顶质膜，在肾脏近曲小管等重吸收过程及尿液浓缩中起重要作用，特别是在髓襻降支细段与逆流倍增机制中发挥关键作用。解除梗阻后较长时间内AQP-1仍未恢复到正常水平，是患儿术后仍有大量稀释尿的原因之一。而如何促进梗阻解除术后AQP-1表达水平的恢复，目前尚不清楚。

EPO是一种人体内源性糖蛋白激素，但近年来许多研究发现EPO对肾脏具有保护作用，能缓解肾脏细胞损伤^[7,8,9,10]。本团队前期研究发现EPO可促进输尿管梗阻-解除梗阻后大鼠肾脏AQP-2 mRNA、蛋白表达及功能恢复，但其能否促进输尿管梗阻-解除梗阻后大鼠肾脏AQP-1表达及功能恢复尚不清楚。

本研究通过制作BUO幼鼠模型，来模拟临床上手术解除梗阻后的患儿，在梗阻解除后给予中等剂量的EPO干预^[11]，结果发现BUO 24 h后，肾脏AQP-1表达水平显著下调；BUO-R组解除梗阻3 d后AQP-1表达水平已开始升高，解除梗阻7 d后蛋白水平相比之前增加，但增加不够显著；而BUO-R＋EPO组解除梗阻3 d后AQP-1表达水平显著增加，解除梗阻7 d增加更显著，虽未达到正常水平，但已接近。本研究对实验幼鼠24 h饮水量和尿液量以及尿液渗透压进行观察记录，发现EPO能够减轻大量稀释尿液的产生，并提高尿液渗透压。同时对各组幼鼠血浆渗透压、Cr、BUN进行检测，发现BUO-R＋EPO组各项指标显著低于BUO-R组。以上结果说明。EPO不仅能够促进BUO-R幼鼠肾脏AQP-1表达的恢复，而且能促进肾脏功能的恢复。

本研究通过免疫组织化学、Western blot测定蛋白表达，同时记录饮水量、尿液量，测定尿液渗透压、血浆渗透压、肾功能，结合各项指标对BUO幼鼠肾脏AQP-1表达及功能变化进行评价，相对较全面，可信度更高。本研究发现EPO能够促进BUO-R幼鼠肾脏AQP-1表达及肾功能的恢复，但其具体作用机制尚不完全了解，推测外源性EPO可能通过缓解肾脏细胞负担、激活三磷酸酰肌醇蛋白激酶/蛋白激酶B信号通路^[11]等方式促进BUO-R幼鼠肾脏AQP-1表达的恢复，有待进一步研究。此外有无不良反应仍未明确，应用于临床上肾积水患儿的治疗仍需长时间的探索，但有可能成为一种促进肾积水肾脏损伤恢复的新方法。

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参考文献

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贡献者信息

郭曦

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

冯锦锦

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

闫少华

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

文一博

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

文建国

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

通信作者

文建国

郑州大学第一附属医院泌尿外科&

小儿尿动力中心　450003

Email：wenjg@hotmail.com

关键词

促红细胞生成素; 双侧输尿管梗阻; 肾脏; 水通道蛋白-1;

基金项目

国家自然科学基金（81370869）

利益冲突

利益冲突　所有作者均声明不存在利益冲突

历史

出版日期：2019-03-05

收稿日期：2018-02-02

本文编辑

单卫华

Urogenital System Disease

Effect of erythropoietin on the expression and function of renal aquaporin-1 after release of bilateral ureter obstruction in young rats

Xi Guo, Jinjin Feng, Shaohua Yan, Yibo Wen, Jianguo Wen

Published 2019-03-05

Cite as Chin J Appl Clin Pediatr, 2019, 34(5): 347-351. DOI: 10.3760/cma.j.issn.2095-428X.2019.05.007

Abstract

Objective

To investigate the effect of erythropoietin(EPO)on the expression and function of aquaporin-1(AQP-1)in the kidney of young male SD rats after release of bilateral ureter obstruction(BUO-R).

Methods

Forty-eight young SD rats were randomly divided into bilateral ureteral complete obstruction(BUO) group (n=6), BUO-R group (n=12), BUO-R+ EPO group (n=12) and Sham group (n=18). The BUO model was built through bilateral ureteral ligation.BUO group were killed after 24 h, and BUO-R group and BUO-R+ EPO group were relieved after obstruction of 24 h. EPO(500 U/kg)was given to BUO-R+ EPO rats at 1 h after release of BUO, and then repeated 1 d, 3 d and 5 d thereafter and the same volume of 9 g/L saline was simultaneously given to BUO-R rats.The Sham group was prepared in parallel by laparotomy and free dissection of bilateral ureters but not ligated, both side kidneys and blood samples were collected on 3 d and 7 d(24 h, 3 d, 7 d for Sham group) after release of BUO.The urine samples were collected by using metabolic cage before death.The plasma osmotic pressure, creatinine(Cr) and urea nitrogen(BUN) in the plasma of young rats were detected.The expression of AQP-1 protein in all groups of kidney tissues was detected by adopting immunohistochemistry and Western blot.

Results

On day 3 after release of BUO, 24 h water intake and urine volume of BUO-R+ EPO group were higher than those of Sham group, but lower than those of BUO group(P<0.05), the urine osmotic pressure of BUO-R+ EPO group was higher than that of BUO group, but lower than that of Sham group (P<0.05), while plasma osmotic pressure, Cr and BUN of BUO-R+ EPO group were higher than those of Sham group, but lower than those of BUO group(P<0.05), and they were all of lower than BUO group (P<0.05). On day 7 after release of BUO, there was no obvious change in Sham group, and the indexes of BUO-R group and BUO-R+ EPO group gradually recovered, but they still did not reach the normal level (P<0.05). The difference between BUO-R group and BUO-R+ EPO group was statistically significant (P<0.05). The immunohistochemical results showed that the expression of AQP-1 in collecting duct in BUO group was significantly down-regulated compared with that in Sham group, whereas it was slightly weaker in BUO-R group and BUO-R+ EPO group than that of Sham group (P<0.05). Compared with 3 days after release of BUO, the staining intensity of BUO-R+ EPO group and BUO-R group was enhanced, but still lower than that of the Sham group.These results were further confirmed by adopting Western blot, and BUO group was also the lowest of the four groups, and BUO-R+ EPO group was higher than that of BUO group, but lower than that of Sham group (P<0.05).

Conclusion

EPO can promote not only the recovery of AQP-1 protein expression but also the recovery of renal function in young BUO-R rats.

Key words:

Erythropoietin; Ureter obstruction; Aquaporin-1; Kidney

Contributor Information

Xi Guo

Department of Urology and Pediatric Urodynamic Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450003, China

Jinjin Feng

Shaohua Yan

Yibo Wen

Jianguo Wen

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