α-促黑素对全肾缺血再灌注模型大鼠的作用

doi:10.3969/j.issn.2095-4344.2015.27.026

摘要/Abstract

摘要：

背景：肾脏缺血再灌注常合并肾脏及肺脏的急性损伤，且角质细胞生长因子受体及钠通道蛋白在缺血再灌注致急性肾、肺损伤中的表达变化及α-促黑素的治疗作用有待于进一步观察及研究。
目的：探索全肾缺血再灌注模型大鼠中角质细胞生长因子受体、钠通道蛋白的表达以及α-促黑素的治疗作用。
方法：选择健康雄性SD大鼠30只按随机数字表法等分为对照组、缺血再灌注造模组和α-促黑素干预组。缺血再灌注造模组和α-促黑素组大鼠通过肾动脉结扎 30 min 建立全肾缺血/再灌注模型，对照组大鼠仅暴露肾动脉不结扎。α-促黑素干预组大鼠于造模前30 min腹腔注射α-促黑素(0.25 mg/kg)进行干预，缺血再灌注造模组大鼠注射等量(4 mL)生理盐水。
结果与结论：与对照组相比，缺血再灌注造模组与α-促黑素干预组大鼠肾、肺组织含水量明显升高，角质细胞生长因子受体、钠通道蛋白的表达明显下降(P < 0.05)；与缺血再灌注造模组相比，α-促黑素干预组大鼠肾、肺组织含水量明显减少，而角质细胞生长因子受体、钠通道蛋白mRNA及蛋白的表达明显升高(P < 0.05)，且大鼠肾、肺组织充血水肿明显减轻。提示肾缺血再灌注后，角质细胞生长因子受体、钠通道蛋白的表达变化与肾、肺充血水肿等损伤一致，而α-促黑素可以增加角质细胞生长因子受体、钠通道蛋白的表达水平，减轻肾及肺组织的损伤，发挥一定的保护作用。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

关键词: 实验动物, 泌尿系统损伤动物模型, 组织工程, 肾脏, 缺血再灌注, 肺损伤, 急性损伤, 角质细胞生长因子受体, 钠通道蛋白, α-促黑素, 国家自然科学基金

Abstract:

BACKGROUND: Kidney ischemia-reperfusion injury often combines with acute kidney and lung injury. The expression of cutin cell growth factor receptor (KGFR) and alpha sodium channel protein (α-ENaC) in kidney and lung after ischemia-reperfusion injury and the protective effect of α-melanocyte require further observation and research.
OBJECTIVE: To explore the therapeutic effect of α-melanocyte on the expressions of KGFR and α-ENaC in rat models of ischemia-reperfusion injury.
METHODS: A total of 30 healthy male Sprague-Dawley rats were randomly divided into control group, ischemia-reperfusion group and α-MSH group. Models of renal ischemia-reperfusion injury were established by 30-minute ligation of renal artery in the ischemia-reperfusion and α-melanocyte groups. Rats in the control group were only used to expose the renal artery, no ligation. Rats in the α-melanocyte group were intraperitoneally injected with α-melanocyte (0.25 mg/kg) at 30 minutes before model establishment. Rats in the ischemia-reperfusion group were injected with 4 mL of physiological saline.
RESULTS AND CONCLUSION: Compared with control group, water content of kidney and lung increased significantly in rats of ischemia-reperfusion group and a-MSH group, while the levels of KGFR and α-ENaC of kidney and lung in rats were lower (P < 0.05). Compared with the ischemia-reperfusion group, water content of kidney and lung in rats of a-MSH group decreased significantly, while the levels of KGFR and α-ENaC of kidney and lung increased gradually (P < 0.05). Moreover, edema was significantly lessened in the rat kidney and lung. Results confirmed that after renal ischemia-reperfusion injury, KGFR and α-ENaC expression was consistent to the kidney and lung injury. α-MSH could increase the protein and mRNA expression of KGFR and α-ENaC in kidney and lung of rats, reduce the kidney and lung injury, and exert a certain protective effect.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

中图分类号:

R318

姜燕，张仲义，徐岩，刘雪梅. α-促黑素对全肾缺血再灌注模型大鼠的作用[J]. 中国组织工程研究, 2015, 19(27): 4405-4411.

Jiang Yan, Zhang Zhong-yi, Xu Yan, Liu Xue-mei. Effect of alpha-melanocyte in rat models of renal ischemia-reperfusion injury [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(27): 4405-4411.

图/表（结果） 6

Quantitative analysis of experimental animals
During the injection of drugs, body mass, food intake and color of all rats had no obvious change and awake in 45 minutes, allowed free access to water in 1 hour. A total of 30 rats entered the experiment at beginning. It was divided into control group (n=10), ischemia-reperfusion group group (n=10), α-MSH group (n=10). During experimental process, one rat in ischemia-reperfusion group was dead because of misoperation and all the rats of control group and α-MSH group survived. 29 rats were sacrificed at 24 hours after reperfusion. Flowchart of model establishment is listed in Figure 1.

Effect of α-MSH on urine, kidney function and water content of rat kidney and lung after ischemia-reperfusion injury
The urine volume in ischemia-reperfusion group rats was higher than that in the control group and α-MSH group
(P < 0.05). The urine osmotic pressure in the ischemia-reperfusion group was lower than that in the control group and α-MSH group (P < 0.05). The urea nitrogen and creatinine levels in the ischemia-reperfusion group were higher than in the control group and α-MSH group (P < 0.05). There was not obvious change in the urea nitrogen and creatinine in the control group and α-MSH group. Kidney and lung water content of the ischemia- reperfusion group was significantly increased compared with control group and α-MSH group (P < 0.05; Table 1).

Effects of α-MSH on pathological changes caused by ischemia-reperfusion injury in the rat kidney
Control structure complete renal tubule neat to rows was observed by hematoxylin-eosin staining. Kidney in the ischemia-reperfusion group had obvious pathological changes, malpighian tube lumen exfoliated cells and tube type of necrosis, interstitial hyperemia edema, and glomerular change was not significant. In the α-MSH group, tube type was found. In the ischemia-reperfusion group, dead cells reduced, interstitial hyperemia edema was seen (Figure 2A-C).
Effects of α-MSH on the lung pathological changes caused by ischemia-reperfusion injury in the rat kidney
A control lung volume was moderate, the color pink, and no congestion. In the ischemia-reperfusion group, lung volume increased, subcapsular flake bleeding. Compared with the ischemia-reperfusion group, the renal pathological changes of α-MSH group significantly reduced. Structure of normal lung tissue was observed under light: control group, no inflammatory cell infiltration, capillaries expansion and congestion, alveolar wall thickening. After ischemia- reperfusion, the experimental alveolar structure disordered, alveolar epithelium swelled, capillary expansion congestion, alveolar interstitial and alveolar seepage, inflammatory cells and proteins in the pathological change of acute lung injury. In the α-MSH group, alveolar exudates were obviously reduced; interstitial edema and alveolar structure tended to be normal (Figure 2D-F).

Effects of α-MSH on the KGFR and α-ENaC protein expression change in the kidney and lung of each group after ischemia-reperfusion injury in the rat kidney
In the control group, the expressions of KGFR and α-ENaC protein in the alveolar epithelial cells and renal tubular epithelial cells were strongly positive. Compared with the ischemia-reperfusion group, the KGFR and α-ENaC protein expression was significantly increased in the control group and α-MSH group (P < 0.05; Figure 3, Table 2).

Changes in KGFR and α-ENaC mRNA levels in the kidney and lung of each group
The results of RT-PCR showed that KGFR and α-ENaC mRNA levels were significantly increased in the α-MSH group than in the ischemia-reperfusion group (P < 0.05), and it was lower in the α-MSH group and ischemia- reperfusion group than in the control group (P < 0.05; Figure 4, Table 3).

参考文献

引言

材料方法

Design
A randomized controlled animal experiment.

Time and setting
Experiments were performed at the Experimental Animal Room and Central Laboratory of The Affiliated Hospital of Qingdao University, China from February to December 2013.

Materials
Experimental animals
A total of 30 male specific-pathogen-free Sprague-Dawley rats weighing (210±20) g and aged 10–11 weeks were purchased from Experimental Animal Center of Qingdao Institute for Drug Control (Animal License No. SCXK (Lu) 20090012). Rats were fed with free access to food and water, kept in a dry cage at (23±1) ℃. The experiment was conducted in compliance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the ministry of Science and Technology of China^[14].

Reagents and instruments are listed as follows:
Reagents and instruments	Source
α-MSH	Sigma-Aldrich Co. LLC, USA
KGFR antibodies	Wuhan Boster Biological Engineering Co., Ltd., China
α-ENaC rabbit two resistance	Beijing Boorson Biological Technology Co., Ltd., China
Extraction of RNA and reverse transcription of a full range of products and quantitative PCR amplification reagents	Biological Co., Ltd.
Fluorescence quantitative PCR models ABI7500, PCR primers	Shanghai Sangon, China
7020 biochemical analyzer	Hitachi, Japan

Methods
Experimental groups
According to the method of random Numbers, it was divided into control group (n=10), ischemia-reperfusion group (n=10), and α-MSH group (n=10). The experimental rats received preoperative fasting for 12 hours and free drinking water.

Establishment of kidney ischemia-reperfusion model and α-MSH intervention
The experimental rats received preoperative fasting 12 hours and free drinking water. At 30 minutes before the operation, rats were intraperitoneally injected with α-MSH in the α-MSH group 0.25 mg/kg. The physiological saline was added to 4 mL. Rats in the ischemia-reperfusion group were intraperitoneally injected with 4 mL physiological saline. Animal in each group was given 10% chloral hydrate (300 mg/kg) for abdomen anesthesia at 30 minutes after treatment. In the control group, the center of the abdomen was cut. No ligation was conducted after separating renal arteries and veins. In the ischemia-reperfusion and α-MSH groups, ligation was performed after the separation of renal arteries and veins. 30 minutes later, the clamp was loosening. The abdominal cavity was closed after flushing with 37 ℃ saline.

Specimen collection
After model establishment, urine specimen was obtained and urine output was recorded. At 24 hours after blood samples, the rats were executed, and then bilateral kidneys and lungs were fully exposed. The right kidney and right lung tissues were separated, and were used for the determination of moisture content, hematoxylin-eosin staining and immunohistochemical staining. The left kidney and lung tissues were treated with liquid nitrogen and frozen at -80 ℃ in a refrigerator using for RT-RCR.

Detection of urine and serum index
The levels of urine and urine osmotic pressure of all rats were measured. Blood serum 2 mL was centrifuged at 3 500 r/min in a centrifuge for 10 minutes, stored at -20 ℃ in a freezer. Creatinine and urea nitrogen were detected with a biochemical analyzer.

Water content detection in the kidney and lung
The left kidney and left lung were obtained after taking blood and washed with four degrees of precooling saline. The lower right lung and kidney tissues were taken to determine moisture content at the lower right tissue.

Pathological detection of kidney and lung tissue
Upper right lung and kidney tissues were obtained and fixed in 4% formaldehyde in conventional medium for 24 hours, followed by paraffin embedding. Samples were sliced into 5 μm thick sections for hematoxylin-eosin staining. The pathological changes in the kidney and lung were observed.

Immunohistochemical detection of KGFR and α-ENaC expression
5 μm thick paraffin sections were seeded on the slide at 60 ℃ overnight, dewaxed, put into the water, treated with 1% methanol, hydrogen peroxide at room temperature for 10 minutes, washed with PBS, followed by antigen retrieval at room temperature for 10 minutes. After washed with PBS, sections were incubated with normal goat serum for 20 minutes at room temperature. After shaking off redundant liquid, the first antibody was added at 4 ℃ overnight. After washing with PBS, biotin second antibody (IgG) was added at 37 ℃ for 20 minutes. Sections were washed three times with PBS, incubated with horseradish peroxidase avidin working fluid at 37 ℃ for 20 minutes, washed three times with PBS, visualized with 3,3'-diaminobenzidine for 6 minutes, and washed with running water. Computer Image processing system (CMOS push around company, Japan) and special software (American Media Cybernetics Corporation Image - Pro Plus) were used. Five horizons were measured in each group. The relative content of positive reactant (grey value and the area) was calculated by a computer.

Real time-PCR for of KGFR and α-ENaC expression
The right lung and right kidney were crushed. 1 mL of RLT was added. 5 μL RNA was obtained, followed by electrophoresis on 1% agarose gel. The residue of RNA genome DNA was digested using DNase I kits.

A kit for reverse transcription was used, with the first chain synthesis reaction system 20 μL, reaction conditions of
65 ℃ for 5 minutes, 37 ℃ for 50 minutes, 70 ℃ heat preservation for 10 minutes. RT-PCR reaction system
20 μL contains 2 × ULtraSYBR Mixture 10 μL, up, down 0.4 μL primers, DNA template 2 μL added in sterilized distilled water to 20 μL. The sample DNA was added, diluted and amplified with target gene primers and internal primers. At the same time, melting curve analysis was conducted at 60-95 ℃; screened primers had been taken in accordance with the principle of amplification efficiency.

Main outcome measures
We measured α-MSH effects on The KGFR and α-ENaC in the kidney and lung after renal ischemia-reperfusion injury.

Statistical analysis
Measurement data are expressed as the mean ± SD, according to statistical analysis between groups compared with SPSS 19.0 software using single factor analysis of variance, comparing two least significant difference-t test. A value of P < 0.05 was considered statistically significant.

讨论

Kidney ischemia-reperfusion injury is an important factor leading to acute renal failure[15-17], although dialysis technology continues to improve, but the kidney ischemia-reperfusion mortality, have inseparable relations with other viscera damage. Patients with renal ischemia-reperfusion injury often complicating acute lung injury, results in greatly increases mortality, close to 80% in the intensive care unit[18-20]. Due to the large vascular network system, lung is the easiest damaged organ. In recent years, the relationship between kidney and lung is focused deeply by people.

α-ENaC is a water Na related membrane transporters. Each organ water transport mainly has two ways[21]. One is by the passive transport of AQP, depending on the pressure in the cell and the number of capillary pressure and water channel protein. Second, with sodium active transport, this system consists of ENaC, Na+-K+ atpase and AQP form, so the reduction of ENaC protein can result in kidney and lung edema, and aggravate damage[22-24]. Previous studies mainly concentrated on ischemia-reperfusion injury such as calcium overload, reactive oxygen species. Currently, many studies have shown that ENaC plays a very important role in cerebral ischemia-reperfusion injury and lung injury[25].

KGFR is a kind of the fibroblast growth factor receptor-2 glycine kinase gene encoding, KGFR specifically expressed in the kidney and lung, such as the surface membrane, is corneous cell growth factor receptor, whose combination can play a role in epithelial cell proliferation and differentiation and liquid removal[26-27]. KGFR structure contains cells, across the membrane, intracellular paragraphs. KGF expression is limited to mesenchymal cells, whereas KGFR expression in epithelial cells[28]. Previous studies found that KGF combined with KGFR induction of Na+, K+ atpase ion channels-a-1 subunits mRNA expression, thus increasing number of Na+, K+ ion channels, promoted cell liquid removal ability, and realized the removal of the liquid in the cells of acute lung injury[29-30]. In addition, KGFR combined with KGF could promote epithelial cell proliferation, differentiation and damage[31-34]. Therefore, the epithelial cell surface expression of KGFR number is very important in the clinical treatment of ischemia reperfusion, which causes acute lung injury.

In this experiment, through bilateral renal arteriovenous separation, double renal arteries were clipped for 30 minutes and blood vessel was open to establish models of renal ischemia-reperfusion injury. Visible to the naked eye color changed from red to dark red kidney and turned bright red, the process of preparation of ischemia reperfusion model is successful. In the ischemia-reperfusion group, urine was markedly increased, urine osmotic pressure reduced, and serum creatinine, blood urea nitrogen elevated, such as impaired renal function. Main pathological changes after renal ischemia-reperfusion injury were renal collecting duct cavity exfoliated cells and tube type of necrosis, interstitial hyperemia edema. Alveolar epithelial lung injury is the main pathology of pulmonary edema, swelling, and wider alveolar walls or expansion of capillary hyperemia, alveolar interstitial and alveolar inflammatory cells and proteins. At 24 hours after the renal ischemia-reperfusion injury, KGFR and α-ENaC expression was decreased significantly in the rat kidney and lung compared with control group, which may be a kidney ischemia-reperfusion injury and lung injury caused by one of the reasons.

α-MSH is a kind of neuroendocrine hormones, consists of 13 amino acid residues, whose precursor is black, the original (Proopiomelanocortin, POMC)[35]. Research shows that malenocortins plays a positive role in the brain with cardiac ischemic injury[12-13, 36-37]. In recent years, numerous studies have confirmed that α-melanocyte plays a significant protective effect in the ischemia-reperfusion injury[38-39], whose mechanism may be related to the antioxidant, control inflammation, restrain apoptosis, stimulate the protective formation. However, it does not report whether it plays a role in reducing water retention of sodium in the kidney and lung. The study found that KGFR and α-ENaC expression was decreased in rat kidney and lung of α-MSH group, but increased significantly in the ischemia-reperfusion group. Pathological changes in the kidney and lung were positively correlated with α-MSH. It suggests that α-MSH can be expressed by increasing KGFR and α-ENaC and alleviate kidney ischemia-reperfusion and lung damage.

Changes in KGFR and α-ENaC expression in the kidney caused by ischemia-reperfusion lung injury and the comparison of the degree of α-MSH effect between them are still needed further research.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

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